Treatment of sirtuin (sirt) related diseases by inhibition of natural antisense transcript to a sirtuin (sirt)

ABSTRACT

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of a Sirtuin (SIRT), in particular, by targeting natural antisense polynucleotides of a Sirtuin (SIRT). The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of Sirtuins (SIRT)s.

The present application claims the priority of U.S. provisional patentapplication No. 61/228,392 filed Jul. 24, 2009, U.S. provisional patentapplication No. 61/259,072, filed Nov. 6, 2009, PCT patent applicationNo. PCT/US09166445 filed Dec. 2, 2009, PCT patent application No.PCT/US10/26119 filed Mar. 3, 2010 and U.S. provisional patentapplication No. 61/330,427 filed May 3, 2010.

FIELD OF TILE INVENTION

Embodiments of the invention comprise oligonucleotides modulatingexpression and/or function of a Sirloin (SERF) and associated molecules.

BACKGROUND

DNA-RNA and RNA-RNA hybridization are important to many aspects ofnucleic acid function including DNA replication, transcription, andtranslation. Hybridization is also central to a variety of technologiesthat either detect a particular nucleic acid or alter its expression.Antisense nucleotides, for example, disrupt gene expression byhybridizing to target RNA, thereby interfering with RNA splicing,transcription, translation, and replication. Antisense DNA has the addedfeature that DNA-RNA hybrids serve as a substrate for digestion byribonuclease H, an activity that is present in most cell types.Antisense molecules can be delivered into cells, as is the case foroligodeoxynucleotides (ODNs), or they can be expressed from endogenousgenes as RNA molecules. The FDA recently approved an antisense drug,VITRAVENE™ (for treatment of cytomegalovirus retinitis), reflecting thatantisense has therapeutic utility.

SUMMARY

In one embodiment, the invention provides methods for inhibiting theaction of a natural antisense transcript by using antisenseoligonucleotide(s) targeted to any region of the natural antisensetranscript resulting in up-regulation of the corresponding sense gene.It is also contemplated herein that inhibition of the natural antisensetranscript can be achieved by siRNA, ribozymes and small molecules,which are considered to be within the scope of the present invention.

One embodiment provides a method of modulating function and/orexpression of a Sirtuin (SIRT) polynucleotide in patient cells ortissues in vivo or in vitro comprising contacting said cells or tissueswith an antisense oligonucleotide 5 to 30 nucleotides in length whereinsaid oligonucleotide has at least 50% sequence identity to a reversecomplement of a polynucleotide comprising 5 to 30 consecutivenucleotides within nucleotides 1 to 1028 of SEQ ID NO: 5 or nucleotides1 to 429 of SEQ ID NO: 6, or nucleotides 1 to 156 of SEQ ID NO: 7 ornucleotides 1 to 593 of SEQ ID NO:8, 1 to 373 of SEQ ID NO: 9, 1 to 1713of SEQ ID NO: 10, 1 to 660 of SEQ ID NO: 11, 1 to 589 of SEQ ID NO: 12,1 to 428 of SEQ ID NO: 13 and 1 to 4441 of SEQ ID NO: 14 therebymodulating function and/or expression of the Sirtuin (SIRT)polynucleotide in patient cells or tissues in vivo or in vitro.

In another embodiment, an oligonucleotide targets a natural antisensesequence of a Sirtuin (SIRT) polynucleotide, for example, nucleotidesset forth in SEQ ID NO: 5 to 14, and any variants, alleles, homologs,mutants, derivatives, fragments and complementary sequences thereto.Examples of antisense oligonucleotides are set forth as SEQ ID NOS: 15to 94.

Another embodiment provides a method of modulating function and/orexpression of a Sirtuin (SIRT) polynucleotide in patient cells ortissues in vivo or in vitro comprising contacting said cells or tissueswith an antisense oligonucleotide 5 to 30 nucleotides in length whereinsaid oligonucleotide has at least 50% sequence identity to a reversecomplement of the an antisense of the Sirtuin (SIRT) polynucleotide;thereby modulating function and/or expression of the Sirtuin (SIRT)polynucleotide in patient cells or tissues in viva or in vitro.

Another embodiment provides a method of modulating function and/orexpression of a Sirtuin (SIRT) polynucleotide in patient cells ortissues in vivo or in vitro comprising contacting said cells or tissueswith an antisense oligonucleotide 5 to 30 nucleotides in length whereinsaid oligonucleotide has at least 50% sequence identity to an antisenseoligonucleotide to a Sirtuin (SIRT) antisense polynucleotide; therebymodulating function and/or expression of the Sirtuin (SIRT)polynucleotide in patient cells or tissues in vivo or in vitro.

In one embodiment, a composition comprises one or more antisenseoligonucleotides which bind to sense and/or antisense Sirtuin (SIRT)polynucleotides.

In another embodiment, the oligonucleotides comprise one or moremodified or substituted nucleotides.

In another embodiment, the oligonucleotides comprise one or moremodified bonds.

In yet another embodiment, the modified nucleotides comprise modifiedbases comprising phosphorothioate, methylphosphonate, peptide nucleicacids, 2′-O-methyl, fluoro- or carbon, methylene or other locked nucleicacid (LNA) molecules. Preferably, the modified nucleotides are lockednucleic acid molecules, including α-L-LNA.

In another embodiment, the oligonucleotides are administered to apatient subcutaneously, intramuscularly, intravenously orintraperitoneally.

In another embodiment, the oligonucleotides are administered in apharmaceutical composition. A treatment regimen comprises administeringthe antisense compounds at least once to patient; however, thistreatment can be modified to include multiple doses over a period oftime. The treatment can be combined with one or more other types oftherapies.

In another embodiment, the oligonucleotides are encapsulated in aliposome or attached to a carrier molecule (e.g. cholesterol, TATpeptide).

Other aspects are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 show Real time PCR results of oligonucleotides designed toSIRT antisense CV396200. The results show that the levels of the SIRT1mRNA in HepG2 cells are significantly increased 48 h after treatmentwith one of the siRNAs designed to sofas (sirtas_(—)5, P=0.01). In thesame samples the levels of sirtas RNA were significantly decreased aftertreatment with sirtas_(—)5, but unchanged after treatment withsirtas_(—)6 and sirtas_(—)7, which also had no effect on the SIRT1 mRNAlevels (FIG. 2). sirtas_(—)5, sirtas_(—)6 and sirtas_(—)7 correspond toSEQ ID NOs: 38, 39 and 40 respectively.

FIG. 3 shows results for the oligonucleotide walk across the SIRTantisense. Real time PCR results show that the levels of the SIRT1 mRNAin HepG2 cells are significantly increased 48 h. after treatment withthree of the antisense oligonucleotides designed to sirtas. CUR-0292 toCUR-0309 correspond to SEQ ID NOs: 15 to 32 respectively.

FIGS. 4 and 5 show results for PS, LNA and 2′O Me Modifiedoligonucleotides HepG2 (FIG. 4) and Vero76 (FIG. 5) cells. Real time PCRresults show that the levels of the SUM mRNA in HepG2 cells aresignificantly increased 48 h after treatment with PS, LNA, 2′O Me and2′O Me mixmer designed antisense oligonucleotides to SIRT1 antisense.Levels of SIRT1 mRNA in Veto cells also increased 48 hours aftertreatment with PS and LNA modified antisense oligonucleotides to SIRT1antisense. Bars denoted as CUR-0245, CUR-0736, CUR 0688, CUR-0740 andCUR-0664 correspond to SEQ ID NOs: 33 to 37 respectively.

FIG. 6 shows PCR results of Monkey Fat Biopsies. Real time PCR resultsshow an increase in SIRT1 mRNA levels in fat biopsies from monkeys dosedwith CUR-963, an oligonucleotide designed to SIRT1 antisense CV396200.1.CUR-963 corresponds to SEQ ID NO: 34.

FIG. 7 shows PCR results of primary monkey liver hepatocytes. Real timePCR results show an increase in SIRT1 mRNA levels after treatment withan oligonucleotide against SIRT1 antisense. Bar denoted as CUR-0245corresponds to SEQ ID NO: 33.

FIG. 8 shows results for oligonucleotides designed to SIRT antisenseCV396200. Real Time PCR results show that levels of SIRT1 mRNA in HepG2cells are significantly increased in one of the oligonucleotidesdesigned to SIRT1 antisense CV396200. The bars denoted as CUR-1230,CUR-1231, CUR-1232 and CUR-1233 correspond to SEQ ID NOs: 41 to 44.

FIG. 9 shows results for oligonucleotides designed to SIRT antisenseCV428275. Real Time PCR results show that levels of SIRT1 mRNA in HepG2cells are significantly increased in two of the oligonucleotidesdesigned to SIRT1 antisense CV428275. The bars denoted as CUR-1302,CUR-1304, CURA 303 and CUR-1305 correspond to SEQ ID NOs: 45 to 48.

FIG. 10 shows Real time PCR results. The results show that a significantincrease in slim mRNA levels in HepG2 cells 48 hours after treatmentwith one of the oligonucleotides designed to SIRT antisense BE717453.The bars denoted as CUR-1264, CUR1265 and CUR-1266 correspond to SEQ IDNOs: 49 to 51 respectively.

FIG. 11 shows Real time PCR results. The results show that show that thelevels of the SIRT1 mRNA in HepG2 cells are significantly increased 48 hafter treatment with three of the oligonucleotides designed to SIRT1antisense AV718812. The bars denoted as CUR-1294, CUR-1297, CUR-1295,CUR-1296 and CUR-1298 correspond to SEQ ID NOs: 52 to 56 respectively.

FIG. 12 is a graph of real time PCR results showing the foldchange+standard deviation in SIRT1 mRNA after treatment of HepG2 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real time PCR results show that the levelsof SIRT1 mRNA are significantly increased in HepG2 cells 48 h aftertreatment with two of the oligos designed to SIRT1 antisense AW169958.Bars denoted as CUR-1381, CUR-1382, CUR-4383 and CUR-1384 correspond tosamples treated with SEQ ID NOS: 57 to 60 respectively.

FIG. 13 is a graph of real time PCR results showing the foldchange+standard deviation in SIRT1 mRNA after treatment of 3T3 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real time PCR results show that the levelsof SIRT1 mRNA are significantly increased in 3T3 cells 48 h aftertreatment with three of the oligonucleotides designed to SIRT1 mouseantisense AK044604. Bars denoted as CUR-0949, CUR-0842, CUR-1098 andCUR-1099 correspond to samples treated with SEQ ID NOS: 67, 61, 71 and72 respectively.

FIG. 14 is a graph of real time PCR results showing the foldchange+standard deviation in SIRT1 mRNA after treatment of 3T3 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real time PCR results show that the levelsof SIRT1 mRNA are significantly increased in 3T3 cells 48 h aftertreatment with five of the oligonucleotides designed to SIRT1 mouseantisense AK044604. Bars denoted as CUR-0948, CUR-0949, CUR-0950,CUR-0951, CUR-0846, and CUR-0844 correspond to samples treated with SEQID NOS: 66 to 69, 65 and 63 respectively.

FIG. 15 is a graph of real time PCR results showing the foldchange+standard deviation in SIRT1 mRNA after treatment of 3T3 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real time PCR results show that the levelsof SIRT1 mRNA are significantly increased in HepG2 cells 48 h aftertreatment with two of the oligonucleotides designed to SIRT1 mouseantisense AK044604. Bars denoted as CUR-0842, CUR-0844, and CUR-0845correspond to samples treated with SEQ ID NOS: 61, 63 and 64respectively.

FIG. 16 is a graph of real time PCR results showing the foldchange+standard deviation in SIRT1 mRNA after treatment of 3T3 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real time PCR results show that the levelsof SIRT1 mRNA are significantly increased in HepG2 cells 48 h aftertreatment with two of the oligonucleotides designed to SIRT1 mouseantisense AK044604. Bars denoted as CUR-0843, CUR-0846 correspond tosamples treated with SEQ ID NOS: 62 and 65 respectively.

FIG. 17 is a graph of real time PCR results showing the foldchange+standard deviation in Sirtuin3 mRNA after treatment of HepG2cells with phosphorothioate oligonucleotides introduced usingLipofectamine 2000, as compared to control. RT PCR results show thatsirt3 levels in HepG2 cells are increased 48 hours after treatment withphosphorothioate antisense oligonucleotides designed to sirt3 antisenseHs.683117 (CUR-1545-1550). Bars denoted as CUR-0551, CUR-1552, CUR-1555,CUR-1556, CUR-1553, CUR-1554, CUR-1545, CUR-1546, CUR-1548, CUR-1549,CUR-1550 and CUR-1547, correspond to samples treated with SEQ ID NOS: 73to 84 respectively.

FIG. 18 is a graph of real time PCR results showing the fold changestandard deviation in SIRT6 mRNA after treatment of HepG2 cells withphosphorothioate oligonucleotides introduced using Lipofectamine 2000,as compared to control. Real time PCR results show that the levels ofSIRT6 mRNA in HepG2 cells are significantly increased 48 h aftertreatment with one of the oliogs designed to SIRT6 antisenseNM_(—)133475. Bars denoted as CUR-0873, CUR-0869 to CUR-0871, CUR-0874and CUR-0872, correspond to samples treated with SEQ ID NOS: 85 to 90respectively.

FIG. 19 is a graph of real time PCR results showing the foldchange+standard deviation in SIRT6 mRNA after treatment of HepG2 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real time PCR results show that the levelsof SIRT6 mRNA in HepG2 cells are significantly increased 48 h aftertreatment with one of the oliogs designed to SIRT6 antisense bf772662.Bars denoted as CUR-0878, CUR-0876, CUR-0877 and CUR-0875, correspond tosamples treated with SEQ ID NOS: 91 to 94 respectively.

FIG. 20 is a graph of real time PCR results showing the foldchange+standard deviation in SIRT6 mRNA after treatment of DBS-FCL-1cells with phosphorothioate oligonucleotides introduced usingLipofectamine 2000, as compared to control. Real time PCR results showthat the levels of SIRT6 mRNA in DBS-FCL-1 cells are significantlyincreased 48 h after treatment with two of the oliogs designed to SIRT6antisense bf772662 and one oligo designed to NM_(—)133475. Bars denotedas CUR-0876, CUR-0878, CUR-0875 and CUR-0874, correspond to samplestreated with SEQ ID NOS: 92, 91, 94 and 89 respectively.

Sequence Listing Description

SEQ ID NO: 1: Homo sapiens sirtuin (silent mating type informationregulation 2 homolog) 1 (S. cerevisiae) (SIRT1), mRNA (NCBI AccessionNumber: NM_(—)012238.3)SEQ ID NO: 2: Mus musculus sirtuin 1 (silent mating type informationregulation 2, homolog) 1 (S. cerevisiae) (SIRT1) mRNA (NCBI AccessionNumber: NM_(—)001159589)SEQ ID NO: 3: Homo sapiens sirtuin (silent mating type informationregulation 2 homolog) 3 (S. cerevisiae) (SIRT3), transcript variant 1,mRNA (NCBI Accession No.: NM_(—)912239.5).SEQ ID NO: 4: Homo sapiens sirtuin 6 (SIRT6), transcript variant 1, mRNA(NCBI Accession No.: NM_(—)016539).SEQ ID NO: 5: Expanded natural antisense sequence (CV396200-expanded)SEQ ID NO: 6: NaturalAntisense sequence (CV428275)

SEQ ID NO: 7: Natural Antisense Sequence (BE717453) SEQ ID NO: 8:Natural Antisense Sequence (AV718812)

SEQ ID NO: 9: Natural SIRT1 antisense sequence (AW169958)SEQ ID NO: 10 Natural SIRT1 mouse antisense sequence (AK044604)SEQ ID NO: 11: Natural SIRT3 antisense sequence (Hs.683117)SEQ ID NO: 12: Natural SIRT3 antisense sequence (DA645474)SEQ ID NO: 13: Natural SIRT6 antisense sequence (BF772662)SEQ ID NO: 14: Natural SIRT6 antisense sequence (ANKRD24)SEQ ID NOs: 15 to 94: Antisense oligonucleotides, * indicatesphosphothioate bond, +indicates LNA and m indicates 2′O MeSEQ ID NO: 95 to 98—SEQ 95 correspond to the exon 4 of the SIRT1 naturalantisense CV396200, SEQ ID NO: 96, 97 and 98 correspond to the forwardprimer sequence, reverse primer sequence and the reporter sequencerespectively.SEQ ID NO: 99 corresponds to CUR 962, * indicates phosphothioate bondand +indicates LNA.

DETAILED DESCRIPTION

Several aspects of the invention are described below with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. The present invention is not limited by the ordering ofacts or events, as some acts may occur in different orders and/orconcurrently with other acts or events. Furthermore, not all illustratedacts or events are required to implement a methodology in accordancewith the present invention.

All genes, gene names, and gene products disclosed herein are intendedto correspond to homologs from any species for which the compositionsand methods disclosed herein are applicable. Thus, the terms include,but are not limited to genes and gene products from humans and mice. Itis understood that when a gene or gene product from a particular speciesis disclosed, this disclosure is intended to be exemplary only, and isnot to be interpreted as a limitation unless the context in which itappears clearly indicates. Thus, for example, for the genes disclosedherein, which in some embodiments relate to mammalian nucleic acid andamino acid sequences are intended to encompass homologous and/ororthologous genes and gene products from other animals including, butnot limited to other mammals, fish, amphibians, reptiles, and birds. Inembodiments, the genes or nucleic acid sequences are human.

DEFINITIONS

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Aswed herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively. “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

As used herein, the term “mRNA” means the presently known mRNAtranscript(s) of a targeted gene, and any further transcripts which maybe elucidated.

By “antisense oligonucleotides” or “antisense compound” is meant an RNAor DNA molecule that binds to another RNA or DNA (target RNA, DNA). Forexample, if it is an RNA oligonucleotide it binds to another RNA targetby means of RNA-RNA interactions and alters the activity of the targetRNA. An antisense oligonucleotide can upregulate or downregulateexpression and/or function of a particular polynucleotide. Thedefinition is meant to include any foreign RNA or DNA molecule which isuseful from a therapeutic, diagnostic, or other viewpoint. Suchmolecules include, for example, antisense RNA or DNA molecules,interference RNA (RNAi), micro RNA, decoy RNA molecules, siRNA,enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA,antisense oligomeric compounds, antisense oligonucleotides, externalguide sequence (EGS) oligonucleotides, alternate splicers, primers,probes, and other oligomeric compounds that hybridize to at least aportion of the target nucleic acid. As such, these compounds may beintroduced in the form of single-stranded, double-stranded, partiallysingle-stranded, or circular oligomeric compounds.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. The term “oligonucleotide”, alsoincludes linear or circular oligomers of natural and/or modifiedmonomers or linkages, including deoxyribonucleosides, ribonucleosides,substituted and alpha-anomeric forms thereof, peptide nucleic acids(PNA), locked nucleic acids (LIMA), phosphorothioate, methylphosphonate,and the like. Oligonucleotides are capable of specifically binding to atarget poly-nucleotide by way of a regular pattern of monomer-to-monomerinteractions, such as Watson-Crick type of base pairing, Hoögsteen orreverse Hoögsteen types of base pairing, or the like.

The oligonucleotide may be “chimeric”, that is, composed of differentregions. In the context of this invention “chimeric” compounds areoligonucleotides, which contain two or more chemical regions, forexample, DNA region(s), RNA region(s), PNA region(s) etc. Each chemicalregion is made up of at least one monomer unit, i.e., a nucleotide inthe case of an oligonucleotides compound. These oligonucleotidestypically comprise at least one region wherein the oligonucleotide ismodified in order to exhibit one or more desired properties. The desiredproperties of the oligonucleotide include, but are not limited, forexample, to increased resistance to nuclease degradation, increasedcellular uptake, and/or increased binding affinity for the targetnucleic acid. Different regions of the oligonucleotide may thereforehave different properties. The chimeric oligonucleotides of the presentinvention can be formed as mixed structures of two or moreoligonucleotides, modified oligonucleotides, oligonucleotides and/oroligonucleotide analogs as described above.

The oligonucleotide can be composed of regions that can be linked in“register” that is, when the monomers are linked consecutively, as innative DNA, or linked via spacers. The spacers are intended toconstitute a covalent “bridge” between the regions and have in cases alength not exceeding about 100 carbon atoms. The spacers may carrydifferent functionalities, for example, having positive or negativecharge, carry special nucleic acid binding properties (intcrealators,groove binders, toxins, fluorophors etc.), being lipophilic, inducingspecial secondary structures like, for example, alanine containingpeptides that induce alpha-helices.

As used herein “Sirtuins (SIRT)s” are inclusive of all family members,mutants, alleles, fragments, species, coding and noncoding sequences,sense and antisense polynueleotide strands, etc.

As used herein, the words Sirtuin1, SIRT1, sirtuin, silent mating typeinformation regulation 2 homolog 1, hSIR2, hSIRT1, NAD-dependentdeacetylase sirtuin-1, SIR2L1, SIR2-like protein 1, are considered thesame in the literature and are used interchangeably in the presentapplication.

As used herein, the words ‘Sirtuin 3’, Sirtuin3, Sirtuin-3, SIRT3,SIRT-3, hSIRT3, NAD-dependent deacetylase sirtuin-3, mitochondrial,SIR2L3, SIR2-like protein 3 are used interchangeably in the presentapplication.

As used herein, the words ‘Sirtuin 6’, Sirtuin6, Sirtuin-6, SIRT6,SIRT-6, NAD-dependent deacetylase sirtuin-6, SIR2L6, SIR2-like protein 6are considered the same in the literature and are used interchangeablyin the present application.

As used herein, the term “oligonucleotide specific for” or“oligonucleotide which targets” refers to an oligonucleotide having asequence (i) capable of forming a stable complex with a portion of thetargeted gene, or (ii) capable of forming a stable duplex with a portionof a mRNA transcript of the targeted gene. Stability of the complexesand duplexes can be determined by theoretical calculations and/or invitro assays. Exemplary assays for determining stability ofhybridization complexes and duplexes are described in the Examplesbelow.

As used herein, the term “target nucleic acid” encompasses DNA, RNA(comprising premRNA and mRNA) transcribed from such DNA, and also cDNAderived from such RNA, coding, noncoding sequences, sense or antisensepolynucleotides. The specific hybridization of an oligomeric compoundwith its target nucleic acid interferes with the normal function of thenucleic acid. This modulation of function of a target nucleic acid bycompounds, which specifically hybridize to it, is generally referred toas “antisense”. The functions of DNA to be interfered include, forexample, replication and transcription. The functions of RNA to beinterfered, include all vital functions such as, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in or facilitatedby the RNA. The overall effect of such interference with target nucleicacid function is modulation of the expression of an encoded product oroligonucleotides.

RNA interference “RNAi” is mediated by double stranded RNA (dsRNA)molecules that have sequence-specific homology to their “target” nucleicacid sequences. In certain embodiments of the present invention, themediators are 5-25 nucleotide “small interfering” RNA duplexes (siRNAs).The siRNAs are derived from the processing of dsRNA by an RNase enzymeknown as Dicer. siRNA duplex products are recruited into a multi-proteinsiRNA complex termed RISC (RNA Induced Silencing Complex). Withoutwishing to be bound by any particular theory, a RISC is then believed tobe guided to a target nucleic acid (suitably mRNA), where the siRNAduplex interacts in a sequence-specific way to mediate cleavage in acatalytic fashion. Small interfering RNAs that can be used in accordancewith the present invention can be synthesized and used according toprocedures that are well known in the art and that will be familiar tothe ordinarily skilled artisan. Small interfering RNAs for use in themethods of the present invention suitably comprise between about 1 toabout 50 nucleotides (nt). In examples of non limiting embodiments,siRNAs can comprise about 5 to about 40 nt, about 5 to about 30 nt,about 10 to about 30 nt, about 15 to about 25 nt, or about 20-25nucleotides.

Selection of appropriate oligonucleotides is facilitated by usingcomputer programs that automatically align nucleic acid sequences andindicate regions of identity or homology. Such programs are used tocompare nucleic acid sequences obtained, for example, by searchingdatabases such as GenBank or by sequencing PCR products. Comparison ofnucleic acid sequences from a range of species allows the selection ofnucleic acid sequences that display an appropriate degree of identitybetween species. In the case of genes that have not been sequenced,Southern blots are performed to allow a determination of the degree ofidentity between genes in target species and other species. Byperforming Southern blots at varying degrees of stringency, as is wellknown in the art, it is possible to obtain an approximate measure ofidentity. These procedures allow the selection of oligonucleotides thatexhibit a high degree of complementarity to target nucleic acidsequences in a subject to be controlled and a lower degree ofcomplementarity to corresponding nucleic acid sequences in otherspecies. One skilled in the art will realize that there is considerablelatitude in selecting appropriate regions of genes for use in thepresent invention.

By “enzymatic RNA” is meant an RNA molecule with enzymatic activity.Enzymatic nucleic acids (ribozymes) act by first binding to a targetRNA. Such binding occurs through the target binding portion of anenzymatic nucleic acid which is held in close proximity to an enzymaticportion of the molecule that acts to cleave the target RNA. Thus, theenzymatic nucleic acid first recognizes and then binds a target RNAthrough base pairing, and once bound to the correct site, actsenzymatitally to cut the target RNA.

By “decoy RNA” is meant an RNA molecule that mimics the natural bindingdomain for a ligand. The decoy RNA therefore competes with naturalbinding target for the binding of a specific ligand. For example, it hasbeen shown that over-expression of HIV trans-activation response (TAR)RNA can act as a “decoy” and efficiently binds HIV at protein, therebypreventing it from binding to TAR sequences encoded in the HIV RNA. Thisis meant to be a specific example. Those in the art will recognize thatthis is but one example, and other embodiments can be readily generatedusing techniques generally known in the art.

As used herein, the term “monomers” typically indicates monomers linkedby phosphodiester bonds or analogs thereof to form oligonucleotidesranging in size from a few monomeric units, e.g., from about 3-4, toabout several hundreds of monomeric units. Analogs of phosphodiesterlinkages include: phosphorothioate, phosphorodithioate,methylphosphomates, phosphoroselenoate, phosphoramidate, and the like,as more fully described below.

The term “nucleotide” covers naturally occurring nucleotides as well asnonnaturally occurring nucleotides. It should be clear to the personskilled in the art that various nucleotides which previously have beenconsidered “non-naturally occurring” have subsequently been found innature. Thus, “nucleotides” includes not only the known purine andpyrimidine heterocycles-containing molecules, but also heterocyclicanalogues and tautomers thereof illustrative examples of other types ofnucleotides are molecules containing adenine, guanine, thymine,cytosine, uracil, purine, xanthine, diaminopurine,8-oxo-N6-methyladenine, 7-deazaxanthine, deazaguanine,N4,N4-ethanocytosin, N6,N6-ethano-2,6-diaminopurine, 5-methylcytosine,5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine,isoguanin, inosine and the “non-naturally occurring” nucleotidesdescribed in U.S. Pat. No. 5,432,272, The term “nucleotide” is intendedto cover every and all of these examples as well as analogues andtautomers thereof. Especially interesting nucleotides are thosecontaining adenine, guanine, thymine, cytosine, and uracil, which areconsidered as the naturally occurring nucleotides in relation, totherapeutic and diagnostic application in humans. Nucleotides includethe natural 2′-deoxy and 2′-hydroxyl sugars, e.g., as described inKomberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco,1992) as well as their analogs.

“Analogs” in reference to nucleotides includes synthetic nucleotideshaving modified base moieties and/or modified sugar moieties. Suchanalogs include synthetic nucleotides designed to enhance bindingproperties, e.g., duplex or triplex stability, specificity, or the like.

As used herein, “hybridization” means the pairing of substantiallycomplementary strands of oligomeric compounds. One mechanism of pairinginvolves hydrogen bonding, which may be Watson-Crick, Hoögsteen orreversed Hoögsteen hydrogen bonding, between complementary nucleoside ornucleotide bases (nucleotides) of the strands of oligomeric compounds.For example, adenine and thymine are complementary nucleotides whichpair through the formation of hydrogen bonds. Hybridization can occurunder varying circumstances.

An antisense compound is “specifically hybridizable” when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a modulation of function and/oractivity, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target nucleicacid sequences under conditions in which specific binding is desired,i.e., under physiological conditions in the case of in vivo assays ortherapeutic treatment, and under conditions in which assays areperformed in the case of in vitro assays.

As used herein, the phrase “stringent hybridization conditions” or“stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances and in the context ofthis invention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated. In general, stringent hybridization conditionscomprise low concentrations (<0.15M) of salts with inorganic cationssuch as Na++ or K++(i.e., low ionic strength), temperature higher than20° C.-25° C. below the Tm of the oligomeric compound target sequencecomplex, and the presence of denaturants such as formamide,dimethylformamide, dimethyl sulfoxide, or the detergent sodium dodecylsulfate (SDS). For example, the hybridization rate decreases 1.1% foreach 1% formamide. An example of a high stringency hybridizationcondition is 0.1× sodium chloride-sodium citrate buffer (SSC)/0.1% (w/v)SDS at 60° C. for 30 minutes.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleotides on one or two oligomeric strands. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, said target nucleic acid being a DNA, RNA, oroligonucleotide molecule, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to be acomplementary position. The oligomeric compound and the further DNA,RNA, or oligonucleotide molecule are complementary to each other when asufficient number of complementary positions in each molecule areoccupied by nucleotides which can hydrogen bond with each other. Thus,“specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of precise pairing or complementarityover a sufficient number of nucleotides such that stable and specificbinding occurs between the oligomeric compound and a target nucleicacid.

It is understood in the art that the sequence of an oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure,mismatch or hairpin structure). The oligomeric compounds of the presentinvention comprise at least about 70%, or at least about 75%, or atleast about 80%, or at least about 85%, or at least about 90%, or atleast about 95%, or at least about 99% sequence complementarity to atarget region within the target nucleic acid sequence to which they aretargeted. For example, an antisense compound in which 18 of 20nucleotides of the antisense compound are complementary to a targetregion, and would therefore specifically hybridize, would represent 90percent complementarity. In this example, the remaining noncomplementarynucleotides may be clustered or interspersed with complementarynucleotides and need not be contiguous to each other or to complementarynucleotides. As such, an antisense compound which is 18 nucleotides inlength having 4 (four) noncomplementaty nucleotides which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a tIrgetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PoweiBLAST programs known in the art.Percent homology, sequence identity or complementarity, can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., (1981)2, 482-489).

As used herein, the term “Thermal Melting Point (Tm)” refers to thetemperature, under defined ionic strength, pH, and nucleic acidconcentration, at which 50% of the oligonucleotides complementary to thetarget sequence hybridize to the target sequence at equilibrium.Typically, stringent conditions will be those in which the saltconcentration is at least about 0.01 to 1.0 M Na ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short oligonucleotides (e.g., 10 to 50 nucleotide). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide.

As used herein, “modulation” means either an increase (stimulation) or adecrease (inhibition) in the expression of a gene.

The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to a wild typegene. This definition may also include, for example, “allelic,”“splice,” “species,” or “polymorphic” variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. Of particular utility in the invention are variants of wildtype gene products. Variants may result from at least one mutation sinthe nucleic acid sequence and may result in altered mRNAs or inpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes that give rise to variants aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

The resulting polypeptides generally will have significant amino acididentity relative to each other. A polymorphic variant is a variation inthe polynucleotide sequence of a particular gene between individuals ofa given species. Polymorphic variants also may encompass “singlenucleotide polymorphisms” (SNPs) or single base mutations in which thepolynucleotide sequence varies by one base. The presence of SNPs may beindicative of, for example, a certain population with a propensity for adisease state, that is susceptibility virus resistance.

Derivative polynucleotides include nucleic acids subjected to chemicalmodification, for example, replacement of hydrogen by an alkyl, acyl, oramino group. Derivatives, e.g., derivative oligonucleotides, maycomprise non-naturally-occurring portions, such as altered sugarmoieties or inter-sugar linkages. Exemplary among these arephosphorothioate and other sulfur containing species which am known inthe art. Derivative nucleic acids may also contain labels, includingradionucleotides, enzymes, fluorescent agents, chemiluminescent agents,chromogenic agents, substrates, cofactors, inhibitors, magneticparticles, and the like.

A “derivative” polypeptide or peptide is one that is modified, forexample, by glycosylation, pegylation, phosphorylation, sulfation,reduction/alkylation, acylation, chemical coupling, or mild formalintreatment. A derivative may also be modified to contain a detectablelabel, either directly or indirectly, including, but not limited to, aradioisotope, fluorescent, and enzyme label.

As used herein, the term “animal” or “patient” is meant to include, forexample, humans, sheep, elks, deer, mule deer, minks, mammals, monkeys,horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chicken,reptiles, fish, insects and arachnids.

“Mammal” covers warm blooded mammals that are typically under medicalcare (e.g., humans and domesticated animals). Examples include feline,canine, equine, bovine, and human, as well as just human.

“Treating” or “treatment” covers the treatment of a disease-state in amammal, and includes: (a) preventing the disease-state from occurring ina mammal, in particular, when such mammal is predisposed to thedisease-state but has not yet been diagnosed as having it; (b)inhibiting the disease-state, e.g., arresting it development; and/or (c)relieving the disease-state, e.g., causing regression of the diseasestate until a desired endpoint is reached. Treating also includes theamelioration of a symptom of a disease (e.g., lessen the pain ordiscomfort), wherein such amelioration may or may not be directlyaffecting the disease (e.g., cause, transmission, expression, etc.).

As used herein, “cancer” refers to all types of cancer or neoplasm ormalignant tumors found in mammals, including, but not limited to:leukemias, lymphomas, melanomas, carcinomas and sarcomas. The cancermanifests itself as a “tumor” or tissue comprising malignant cells ofthe cancer. Examples of tumors include sarcomas and carcinomas such as,but not limited to: fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma. Additional cancers which can be treated by the disclosedcomposition according to the invention include but not limited to, forexample, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma,neuroblastoma, breast cancer, ovarian cancer, lung cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, coloncancer, malignant pancreatic insulanoma, malignant carcinoid, urinarybladder cancer, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tractcancer, malignant hypercalcemia, cervical cancer, endometrial cancer,adrenal cortical cancer, and prostate cancer.

“Neurological disease or disorder” refers to any disease or disorder ofthe nervous system and/or visual system. “Neurological disease ordisorder” include disease or disorders that involve the central nervoussystem (brain, brainstem and cerebellum), the peripheral nervous system(including cranial nerves), and the autonomic nervous system (parts ofwhich are located in both central and peripheral nervous system).Examples of neurological disorders include but are not limited to,headache, stupor and coma, dementia, seizure, sleep disorders, trauma,infections, neoplasms, neuroopthalmology, movement disorders,demyelinating diseases, spinal cord disorders, and disorders ofperipheral nerves, muscle and neuromuscular junctions. Addiction andmental illness, include, but are not limited to, bipolar disorder andschizophrenia, are also included in the definition of neurologicaldisorder. The following is a list of several neurological disorders,symptoms, signs and syndromes that can be treated using compositions andmethods according to the present invention: acquired epileptiformaphasia; acute disseminated encephalomyelitis; adrenoleukodystrophy;age-related macular degeneration; agenesis of the corpus callosum;agnosia; Aicardi syndrome; Alexander disease; Alpers' disease;alternating hemiplegia; Vascular dementia; amyotrophic lateralsclerosis; anencephaly; Angelman syndrome; angiomatosis; anoxia;aphasia; apraxia; arachnoid cysts; arachnoiditis; Anronl-Chiarimalformation; arteriovenous malformation; Asperger syndrome; ataxiatelegiectasia; attention deficit hyperactivity disorder; autism;autonomic dysfunction; back pain; Batten disease; Behcet's disease;Bell's palsy; benign essential blepharospasm; benign focal; amyotrophy;benign intracranial hypertension; Binswanger's disease; blepharospasm;Bloch Sulzberger syndrome; brachial plexus injury; brain abscess; braininjury; brain tumors (including glioblastoma multiforme); spinal tumor;Brown-Sequard syndrome; Canavan disease; carpal tunnel syndrome;causalgia; central pain syndrome; central pontine myclinolysis; cephalicdisorder; cerebral aneurysm; cerebral arteriosclerosis; cerebralatrophy; cerebral gigantism; cerebral palsy; Charcot-Marie-Toothdisease; chemotherapy-induced neuropathy and neuropathic pain; Chiarimalformation; chorea; chronic inflammatory demyelinating polyneuropathy;chronic pain; chronic regional pain syndrome; Coffin Lowry syndrome;coma, including persistent vegetative state; congenital facial diplegia;corticobasal degeneration; cranial arteritis; craniosynostosis;Creutzfeldt-Jakob disease; cumulative trauma disorders; Cushing'ssyndrome; cytomegalic inclusion body disease; cytomegalovirus infection;dancing eyes-dancing feet syndrome; Dandy Walker syndrome; Dawsondisease; De Morsier's syndrome; Dejerine-Klumke palsy; dementia;dermatomyositis; diabetic neuropathy; diffuse sclerosis; dysaulonomia;dysgraphia; dyslexia; dystonias; early infantile epilepticencephalopathy; empty sella syndrome; encephalitis; encephaloceles;encephalotrigeminal angiomatosis; epilepsy; Erb's palsy; essentialtremor, Fabry's disease; Fahr's syndrome; fainting; familial spasticparalysis; febrile seizures; Fisher syndrome; Friedreich's ataxia;fronto-temporal dementia and other “tauopathies”; Gaucher's disease;Gerstmann's syndrome; giant cell arteritis; giant cell inclusiondisease; globoid cell leukodystrophy; Guillain-Barre syndrome;HTLV-1-associated myelopathy; Hallervorden-Spatz disease; head injury;headache; hemifacial spasm; hereditary spastic paraplegia; heredopathiaatactic a polyneuritiformis; herpes zoster oticus; herpes zoster;Hirayama syndrome; HIV associated dementia and neuropathy (alsoneurological manifestations of AIDS); holoprosencephaly; Huntington'sdisease and other polyglutamine repot diseases; hydranencephaly;hydrocephalus; hypercortisolism; hypoxia; immune-mediatedencephalomyelitis; inclusion body myositis; incontinentia pigmenti;infantile phytanic acid storage disease; infantile refsum disease;infantile spasms; inflammatory myopathy; intracranial cyst; intracranialhypertension; Joubert syndrome; Keams-Sayre syndrome; Kennedy diseaseKinsboume syndrome; Klippel Fell syndrome; Krabbe disease;Kugelberg-Welander disease; kuru; Lafora disease; Lambert-Eatonmyasthenic syndrome; Landau-Kleffner syndrome; lateral medullary(Wallenberg) syndrome; learning disabilities; Leigh's disease;Lennox-Gustaut syndrome; Lesch-Nyhan syndrome; leukodystrophy; Lewy bodydementia; Lissencephaly; locked-in syndrome; Lou Gehrig's disease (i.e.,motor neuron disease or amyotrophic lateral sclerosis); lumbar discdisease; Lyme disease-neurological sequelae; Machado-Joseph disease;macrencephaly; megalencephaly; Melkersson-Rosenthal syndrome; Menieresdisease; meningitis; Menkes disease; metachromatic leukodystrophy;microcephaly; migraine; Miller Fisher syndrome; mini-strokes;mitochondrial myopathies; Mobius syndrome; monomelic amyotrophy; motorneuron disease; Moyamoya disease; mucopolysaccharidoses; milti-infantdementia; multifocal motor neuropathy; multiple sclerosis and otherdemyelinating disorders; multiple system atrophy with posturalhypotension; p muscular dystrophy; myasthenia gravis; myelinoclasticdiffuse sclerosis; myoclonic encephalopathy of infants; myoclonus;myopathy; myotonia congenital; narcolepsy; neurofibromatosis;neuroleptic malignant syndrome; neurological manifestations of AIDS;neurological sequelae oflupus; neuromyotonia; neuronal ceroidlipofuscinosis; neuronal migration disorders; Niemann-Pick disease;O'Sullivan-McLeod syndrome; occipital neuralgia; occult spinaldysraphism sequence; Ohtahara syndrome; olivopontocerebellar atrophy;opsoclonus myoclonus; optic neuritis; orthostatic hypotension; overusesyndrome; paresthesia; Neurodegenerative disease or disorder(Parkinson's disease, Huntington's disease, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), dementia, multiple sclerosis andother diseases and disorders associated with neuronal cell death);paramyotonia congenital; paraneoplastic diseases; paroxysmal attacks;Parry Romberg syndrome; Pelizaeus-Merzbacher disease; periodicparalyses; peripheral neuropathy; painful neuropathy and neuropathicpain; persistent vegetative state; pervasive developmental disorders:photic sneeze: reflex; phytanic acid storage disease; Pick's disease;pinched nerve: pituitary tumors; polymyositis; porencephaly; post-poliosyndrome; postherpetic neuralgia; postinfectious encephalomyelitis;postural hypotension; Prader-Willi syndrome; primary lateral sclerosis;prion diseases; progressive hemifacial atrophy; progressivemultifocalleukoencephalopathy; progressive sclerosing poliodystrophy;progressive supranuclear palsy; pseudotumor cerebri; Ramsay-Huntsyndrome (types 1 and 11); Rasmussen's encephalitis; reflex sympatheticdystrophy syndrome; Refsum disease; repetitive motion disorders;repetitive stress injuries; restless legs syndrome;retrovirus-associated myelopathy; Rett syndrome; iReye's syndrome; SaintVitus dance; Sandhoff disease; Schilder's disease; schizencephaly;septo-optic dysplasia; shaken baby syndrome; shingles; Shy-Dragersyndrome; Sjogren's syndrome; sleep apnea; Soto's syndrome; spasticity;spina bifida; spinal cord injury; spinal cord tumors; spinal muscularatrophy; Stiff-Person syndrome; stroke; Sturge-Weber syndrome; subacutesclerosing panencephalitis; subcortical arteriosclerotic encephalopathy;Sydenham chorea; syncope; syringomyelia; tardive dyskinesia; lay-Sachsdisease temporal arteritis; tethered spinal cord syndrome; Thomsendisease; thoracic outlet syndrome; Tic Douloureux; Todd's paralysis;Tourette syndrome; transient ischemic attack; transmissible spongiformencephalopathies; transverse myelitis; traumatic brain injury; tremor;trigeminal neuralgia; tropical spastic paraparesis; tuberous sclerosis;vascular dementia (multi-infarct dementia); vasculitis includingtemporal arteritis; Von Hippel-Lindau disease; Wallenberg's syndrome;Werdnig-Hoffman disease; West syndrome; whiplash; Williams syndrome;Wildon's disease; and Zellweger syndrome.

“Metabolic disease” refers to a wide range of diseases and disorders ofthe endocrine system including, for example, insulin resistance,diabetes, obesity, impaired glucose tolerance, high blood cholesterol,hyperglycemia, dyslipidemia and hyperlipidemia.

An “Inflammation” refers to systemic inflammatory conditions andconditions associated locally with migration and attraction ofmonocytes, leukocytes and/or neutrophils. Examples of inflammationinclude, but are not limited to, Inflammation resulting from infectionwith pathogenic organisms (including grain-positive bacteria,gram-negative bacteria, viruses, fungi, and parasites such as protozoaand helminths), transplant rejection (including rejection of solidorgans such as kidney, liver, heart, lung or cornea, as well asrejection of bone marrow transplants including graft-versus-host disease(GVHD)), or from localized chronic or acute autoimmune or allergicreactions. Autoimmune diseases include acute glomerulonephritis;rheumatoid or reactive arthritis; chronic glornertilonephritis;inflammatory bowel diseases such as Crohn's disease, ulcerative colitisand necrotizing enterocolitis; granulocyte transfusion associatedsyndromes; inflammatory dermatoses such as contact dermatitis, atopicdermatitis, psoriasis; systemic lupus erythematosus (SLE), autoimmunethyroiditis, multiple sclerosis, and some forms of diabetes, or anyother autoimmune state where attack by the subject's own immune systemresults in pathologic tissue destruction. Allergic reactions includeallergic asthma, chronic bronchitis, acute and delayed hypersensitivity.Systemic inflammatory disease states include inflammation associatedwith trauma, burns, reperfusion following ischemic events (e.g.thrombotic events in heart, brain, intestines or peripheral vasculature,including myocardial infarction and stroke), sepsis, ARDS or multipleorgan dysfunction syndrome inflammatory cell recruitment also occurs inatherosclerotic plaques. Inflammation includes, but is not limited to,Non-Hodgkin's lymphoma, Wegener's granulomatosis, Hashimoto'sthyroiditis, hepatocellular carcinoma, thymus atrophy, chronicpancreatitis, rheumatoid arthritis, reactive lymphoid hyperplasia,osteoarthritis, ulcerative colitis, papillary carcinoma, Crohn'sdisease, ulcerative colitis, acute cholecystitis, chronic cholecystitis,cirrhosis, chronic sialadenitis, peritonitis, acute pancreatitis,chronic pancreatitis, chronic Gastritis, adenomyosis, endometriosis,acute cervicitis, chronic cervicitis, lymphoid hyperplasia, multiplesclerosis, hypertrophy secondary to idiopathic thrombocytopenic purpura,primary IgA nephropathy, systemic lupus erythematosus, psoriasis,pulmonary emphysema, chronic pyelonephritis, and chronic cystitis.

A cardiovascular disease or disorder includes those disorders that caneither cause ischemia or are caused by reperfusion of the heart.Examples include, but are not limited to, atherosclerosis, coronaryartery disease, granulomatous myocarditis, chronic myocarditis(non-granulomatous), primary hypertrophic cardiomyopathy, peripheralartery disease (PAD), stroke, angina pectoris, myocardial infarction,cardiovascular tissue damage caused by cardiac arrest, cardiovasculartissue damage caused by cardiac bypass, cardiogenic shock, and relatedconditions that would be known by those of ordinary skill in the art orwhich involve dysfunction of or tissue damage to the heart orvasculature, especially, but not limited to, tissue damage related toSirtuin3 activation. CVS diseases include, but are not limited to,atherosclerosis, granulomatous myocarditis, myocardial infarction,myocardial fibrosis secondary to valvular heart disease, myocardialfibrosis without infarction, primary hypertrophic cardiomyopathy, andchronic myocarditis (non-granulomatous).

Polynucleotide and Oligonucleotide Compositions and Molecules Targets

In one embodiment, the targets comprise nucleic acid sequences of aSirtuin (SIRT), including without limitation sense and/or antisensenoncoding and/or coding sequences associated with a Sirtuin (SIRT).

In one embodiment, the targets comprise nucleic acid sequences of SIRT1,including without limitation sense and/or antisense noncoding and/orcoding sequences associated with SIRT1 gene.

In one embodiment, the targets comprise nucleic acid sequences of SIRT3,including without limitation sense and/or antisense noncoding and/orcoding sequences associated with SIRT3 gene.

In one embodiment, the targets comprise nucleic acid sequences of SIRT6,including without limitation sense and/or antisense noncoding and/orcoding sequences associated with SIRT6 gene.

“SIRT1 protein” refers to a member of the sir2 family of sirtuindeacetylases. In one embodiment, a SIRT1 protein includes yeast Sir2(GenBank Accession No. P53685), C. elegans Sir-2.1 (GenBank AccessionNo. NP.sub.—501912), human SIRT1 (GenBank Accession No. NM.sub.—012238and NP.sub.—036370 (or AF083106))

SIRT1 “Sirtuins” are proteins that include a SIR2 domain, a domaindefined as amino acids sequences that are scored as hits in the Pfamfamily “SIR2”-PF02146 (attached to the Appendix). This family isreferenced in the INTERPRO database as INTERPRO description (entryIPR003000). To identify the presence of a “SIR2” domain in a proteinsequence, and make the determination that a polypeptide or protein ofinterest has a particular profile, the amino acid sequence of theprotein can be searched against the Pfam database of HMMs (e.g., thePfam database, release 9) using the default parameters(http://www.sanger.ac.uk/Software/Pfam/HMM_search). The SIR2 domain isindexed in Pfam as PF02146 and in INTERPRO as INTERPRO description(entry IPR003000). A description of the Pfam database can be found in“The Plain Protein Families Database” Bateman A et al. (2002) NucleicAcids Research 30(1):276-280 and Sonhammer et al. (1997) Proteins28(3):405-420 and a detailed description of HMMs can be found, forexample, in Gribskov et al. (1990) Meth. Enzymol. 183:146-1.59; Gribskovet al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et at(1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) ProteinSci. 2:305-314.

Among the mitochondrial sirtuins, SIRT3 possesses the most robustdeacetylase activity. Indeed, significantly higher levels ofmitochondrial protein acetylation were detected in the livers ofSIRT3-null mice, compared to those of SIRT4 or SIRT5 knockout animals.However, little is known about the physiological role of SIRT3 despitethe fact that a number of SIRT3 substrates and co-precipitating proteinshave been identified: acetyl-CoA synthetase 2, Ku70, FOXO3a, subunit 9of mitochondrial Complex 1 (NDUFA9), glutamate dehydrogenase andisocitrate dehydrogenase 2.

SIRT3 is a major mitochondrial deacetylase. Mitochondrial proteins showhyperacetylation in SIRT3 knockout mice, but not in SIRT4 or SIRT5knockout mice. Acetyl-CoA synthetase 2 (AceCS2), a mitochondrial enzymethat converts acetate into acetyl-CoA, was the first mitochondrialsubstrate of SIRT3 identified, Deacetylation of AceCS2 at lysine 642 bySIRT3 activates acetyl-CoA synthetase activity, providing increasedacetyl-CoA to feed into the tricarboxylic acid cycle. Glutamatedehydrogenase (GDII), another mitochondrial protein involved in energyproduction, is deacetylated by SIRT3. GDH can also be ADP-ribosylated bySIRT4 in turn to decrease its enzyme activity. This indicates that SIRT3could play an important role in cell metabolism. SIRT3 has also beenshown to be involved in selective apoptosis pathways and cell growthcontrol. SIRT3 and SIRT4, but not SIRT5, have been implicated in theNAD+ salvage pathway that regulates the NAT+ level relating to cellsurvival. In addition, variability in the hSIRT3 gene has been linked tohuman longevity.

The Silent information Regulator-2 gene (Sir2) encodes an NAD-dependenthistone deacetylase that links regulation of chromatin, genomicstability, and life span in S. cerevisiae. By promoting chromatinsilencing, Sir2 inhibits transcription at several genetic loci andrepresses recombination at ribosomal DNA (rDNA) repeats. Yeast withmutations in Sir2 have increased genomic instability in the context ofrDNA recombination, which in turn shortens replicative life span—amarker of reproductive aging in this organism. Conversely, extracopiesof Sir2 that suppress rDNA recombination increase replicative life span.These effects of Sir2 suggest paradigms in which genes that promotegenome stabilization through chromatin modulation may be importantcontributors to regulation of organismal life span, aging, andage-related pathology.

Consistent with a conserved role for Sir2 factors in life spanregulation, increased activity of Sir2 proteins in the multicellularorganisms C. elegans and D. melanogaster also increases life span.However, these Sir2 factors may operate through mechanisms that areindependent of genome stabilization, and their physiologic molecularsubstrates are still unclear. In mammals, there are seven Sir2 familymembers, SIRT1-SIRT7. The SIRTs have been of great interest as candidateregulators of mammalian life span and aging-related processes. In thiscontext, several mammalian SIRTs have functions that impact onaging-associated molecular pathways and disease. However, initialstudies of mammalian SIRTs linked these enzymes to biochemical targetsand cellular functions that are distinct from those of. S. cerevisiaeSir2.

The generation of mice deficient for the mammalian SIRT6 Rene revealed apotential role for SIRT6 in linking regulation of life span, chromatin,and genomic stability. In this context, SIRT6 deficiency in mice leadsto dramatically shortened life span and acute degenerative phenotypesthat overlap with pathologies of premature aging. Moreover, SIRT6knockout mouse cells have genomic instability and DNA damagehypersensitivity. In biochemical fractionation assays, SIRT6 proteinassociates preferentially with a chromatin-enriched cellular fraction.Together, these observations suggested that SIRT6 might couple chromatinregulation with DNA repair. However, a physiologic role for SIRT6 insuch a process has not yet been demonstrated.

In some embodiments, antisense oligonucleotides are used to prevent ortreat diseases or disorders associated with Sirtuin (SIRT) familymembers. Exemplary Sirtuin (SIRT) mediated diseases and disorders whichcan be treated with cell/tissues regenerated from stem cells obtainedusing the antisense compounds comprise: cancer (e.g., breast cancer,colorectal cancer, CCL, CML, prostate cancer), a neurodegenerativedisease or disorder (e.g., Alzheimer's Disease (AD), Huntington'sdisease, Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS),Multiple Sclerosis, and disorders caused by polyglutamine aggregation);skeletal muscle disease (e.g., Duchene muscular dystrophy, skeletalmuscle atrophy, Becker's dystrophy, or myotonic dystrophy); a metabolicdisease or disorder (e.g., insulin resistance, diabetes, type 2diabetes, obesity, impaired glucose tolerance, metabolic syndrome,adult-onset diabetes, diabetic nephropathy, hyperglycemia, diabeticnephropathy, Hypercholesterolemia, dyslipidemia hyperlipidemia and anage-related metabolic disease etc.), a disease or disorder associates)with impaired regulation of insulin level, neuropathy (e.g., sensoryneuropathy, autonomies neuropathy, motor neuropathy, retinopathy), adisease or disorder associated with a ketogenic condition, a disease ordisorder associated with impaired energy homeostasis, a disease ordisorder associated with impaired Acetyl-CoA synthetase 2 activity, adisease or disorder associated with metabolic homeostasis, a lipidmetabolism disease or disorder, a disease or disorder associated withimpaired thermogenesis, a disease or disorder associated withmitochondrial dysfunction, neuropathy (e.g., sensory neuropathy,autonomic neuropathy, motor neuropathy, retinopathy), a liver disease(e.g., due to alcohol abuse or hepatitis, fatty liver disease etc.);age-related macular degeneration, bone disease (e.g., osteoporosis), ablood disease (e.g., a leukemia); liver disease (e.g.: due to alcoholabuse or hepatitis); Obesity; bone resorption, age-related maculardegeneration, AIDS related dementia, ALS, Bell's Palsy, atherosclerosis,a cardiac disease (e.g., cardiac dysrhymias, chronic congestive heartfailure, ischemic stroke, coronary artery disease and cardiomyopathy),chronically degenerative disease (e.g., cardiac muscle disease), chronicrenal failure, type 2 diabetes, ulceration, cataract, presbiopia,glomerulonephritis, Guillan-Barre syndrome, hemorrhagic stroke,rheumatoid arthritis, inflammatory bowel disease, SLE, Crohn's disease,osteoarthritis, osteoporosis, Chronic Obstructive Pulmonary Disease(COPP), pneumonia, skin aging, urinary incontinence, a disease ordisorder associated with mitochondrial dysfunction (e.g., mitochondrialmyopathy, encephalopathy, Leber's disease, Leigh encephalopathia,Pearson's disease, lactic acidosis, mitochondrial encephalopathy, lacticacidosis and stroke like symptoms' (MELAS) etc.) and a disease ordisorder associated with neuronal cell death, degenerative syndrome,aging, a disease or disorder associated with telomere dysfunction, adisease or disorder associated with impaired chromatin regulation, adisease or disorder associated with premature cellular senescence, adisease or disorder associated with impared SIRT6 mediated DNA repairand a condition characterized by unwanted cell loss.

In another embodiment, the antisense oligonucleotides modulate thenormal expression and/or normal function of a Sirtuin (SIRT) in patientssuffering from or at risk of developing diseases or disorders associatedwith Sirtuin (SIRT).

In embodiments of the present invention, therapeutic and/or cosmeticregimes and related tailored treatments are provided to subjectsrequiring skin treatments or at risk of developing conditions for whichthey would require skin treatments. Diagnosis can be made, e.g., basedon the subject's SIRT status. A patient's SIRT expression levels in agiven tissue such as skin can be determined by methods known to those ofskill in the an and described elsewhere herein, e.g., by analyzingtissue using PCR or antibody-based detection methods.

A preferred embodiment of the present invention provides a compositionfor skin treatment and/or a cosmetic application comprising SIRTantisense oligonucleotides, e.g., to upregulate expression of SIRT inthe skin. Examples of antisense oligonucleotides are set forth as SEQ IDNOS: 4 to 16. U.S. Pat. No. 7,544,497, “Compositions for manipulatingthe lifespan and stress response of cells and organisms,” incorporatedherein by reference, describes potential cosmetic use for agents thatmodulate Sirtuin activity by reducing the K_(m) of the Sirtuin proteinfor its substrate. In embodiments, cells are treated in vivo with theoligonucleotides of the present invention, to increase cell lifespan orprevent apoptosis. For example, skin can be protected from aging, e.g.,developing wrinkles, by treating skin, e.g., epithelial cells, asdescribed herein. In an exemplary embodiment, skin is contacted with apharmaceutical or cosmetic composition comprising a SIRT antisensecompound as described herein. Exemplary skin afflictions or skinconditions include, disorders or diseases associated with or caused byinflammation, sun damage or natural aging. For example, the compositionsfind utility in the prevention or treatment of contact dermatitis(including irritant contact dermatitis and allergic contact dermatitis),atopic dermatitis (also known as allergic eczema), actinic keratosis,keratinization disorders (including eczema), epidermolysis bullosadiseases (including penfigus), exfoliative dermatitis, seborrheicdermatitis, erythemas (including erythema multiforme and erythemanodosum), damage caused by the sun or other light sources, discoid lupuserythematosus, dermatomyositis, skin cancer and the effects of naturalaging.

Sirtuin has been reported to interfere with dihydrotestosterone-inducedandrogen receptor signaling. (See, e.g., Fu, et al., 2006, “HormonalControl of Androgen Receptor Function through SIRT1 Molecular andCellular Biology 26(21): 8122-8135, incorporated herein by reference.)In embodiments of the present invention, a composition comprising SIRTantisense oligonucleotides, e.g., to upregulate expression of SIRT inthe scalp and inhibit androgen receptor signaling, thereby preventingandrogenetic alopecia (hair loss). In embodiments, a patient sufferingfrom alopecia is administered either a topical or systemic formulation.

In an embodiment, an antisense oligonucleotide described herein isincorporated into a topical formulation containing a topical carrierthat is generally suited to topical drug administration and comprisingany such material known in the art. The topical carrier may be selectedso as to provide the composition in the desired form, e.g., as anointment, lotion, cream, microemulsion, gel, oil, solution, or the like,and may be comprised of a material of either naturally occurring orsynthetic origin. It is preferable that the selected carrier notadversely affect the active agent or other components of the topicalformulation. Examples of suitable topical carriers for use hereininclude water, alcohols and other nontoxic organic solvents, glycerin,mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetableoils, parabens, waxes, and the like. Formulations may be colorless,odorless ointments, lotions, creams, microemulsions and gels.

Antisense oligonucleotides of the invention may be incorporated intoointments, which generally are semisolid preparations which aretypically based on petrolatum or other petroleum derivatives. Thespecific ointment base to be used, as will be appreciated by thoseskilled in the art, is one that will provide for optimum drug delivery,and, preferably, will provide for other desired characteristics as well,e.g., emolliency or the like. As with other carriers or vehicles, anointment base should be inert, stable, nonirritating and nonsensitizing.As explained in Remington's Pharmaceutical Sciences (Mack Pub. Co.),ointment bases may be grouped into four classes: oleaginous bases;emulsifiable bases; emulsion bases; and water-soluble bases. Oleaginousointment bases include, for example, vegetable oils, fats obtained fromanimals, and semisolid hydrocarbons obtained from petrOleum.Emulsifiable ointment bases, also known as absorbent ointment bases,contain little or no water and include, for example, hydroxystearinsulfate, anhydrous lanolin and hydrophilic petrolatum. Emulsion ointmentbases are either water-in-oil (W/O) emulsions or oil-in-water (O/W)emulsions, and include, for example, cetyl alcohol, glycerylmonostearate, lanolin and stearic acid. Exemplary water-soluble ointmentbases are prepared from polyethylene glycols (PEGs) of varying molecularweight (see, e.g., Remington's, supra).

Antisense oligonucleotides of the invention may be incorporated intolotions, which generally are preparations to be applied to the skinsurface without friction, and are typically liquid or semiliquidpreparations in which solid particles, including the active agent, arepresent in a water or alcohol base. Lotions are usually suspensions ofsolids, and may comprise a liquid oily emulsion of the oil-in-watertype. Lotions are preferred formulations for treating large body areas,because of the ease of applying a more fluid composition. It isgenerally necessary that the insoluble matter in a lotion be finelydivided. Lotions will typically contain suspending agents to producebetter dispersions as well as compounds useful far localizing andholding the active agent in contact with the skin, e.g.,methylcellulose, sodium carboxymethylcellulose, or the like. Anexemplary lotion formulation for use in conjunction with the presentmethod contains propylene glycol mixed with a hydrophilic petrolatumsuch as that which may be obtained under the trademark Aquaphor.sup.RTMfrom Beiersdorf, Inc, (Norwalk, Conn.).

Antisense oligonucleotides of the invention may be incorporated intocreams, which generally are viscous liquid or semisolid emulsions,either oil-in-water or water-in-oil. Cream bases are water-washable, andcontain an oil phase, an emulsifier and an aqueous phase. The oil phaseis generally comprised of petrolatum and a fatty alcohol such as cetylor stearyl alcohol; the aqueous phase usually, although not necessarily,exceeds the oil phase in volume, and generally contains a humectant. Theemulsifier in a cream formulation, as explained in Remington's, supra,is generally a nonionic, anionic, cationic or amphoteric surfactantAntisense oligonucleotides of the invention may be incorporated intomicroemulsions, which generally are thermodynamically stable,isotropically clear dispersions of two immiscible liquids, such as oiland water, stabilized by an interfacial film of surfactant molecules(Encyclopedia of Pharmaceutical Technology (New York: Marcel Dekker,1992), volume 9). For the preparation of microemulsions, surfactant(emulsifier), co-surfactant (co-emulsifier), an oil phase and a waterphase are necessary. Suitable surfactants include any surfactants thatare useful in the preparation of emulsions, e.g., emulsifiers that aretypically used in the preparation of creams. The co-surfactant (or“co-emulsifer”) is generally selected from the group of polyglycerolderivatives, glycerol derivatives and fatty alcohols. Preferredemulsifier/co-emulsifier combinations are generally although notnecessarily selected from the group consisting of: glyceryl monostearateand polyoxyethylene stearate; polyethylene glycol and ethylene glycolpalmitostearate; and caprilic and capric triglycerides and oleoylmacrogolglycerides. The water phase includes not only water but also,typically, buffers, glucose, propylene glycol, polyethylene glycols,preferably lower molecular weight polyethylene glycols (e.g., PEG 300and PEG 400), and/or glycerol, and the like, while the oil phase willgenerally comprise, for example, fatty acid esters, modified vegetableoils, silicone oils, mixtures of mono- di- and triglycerides, mono- anddi-esters of PEG (e.g., oleoyl macrogol glycerides), etc.

Antisense oligonucleotides of the invention may be incorporated into gelfomutlations, which generally are semisolid systems consisting of eithersuspensions made up of small inorganic particles (two-phase systems) orlarge organic molecules distributed stibstantially uniformly throughouta carrier liquid (single phase gelS). Single phase gels can be made, forexample, by combining the active agent, a carrier liquid and a suitablegelling agent such as tragacanth (at 2 to 5%), sodium alginate (at2-10%), gelatin (at 2-15%), methylcellulose (at 3-5%), sodiumcarboxymethylcellulose (at 2-5%), carbomer (at 0.3-5%) or polyvinylalcohol (at 10-20%) together and mixing until a characteristic semisolidproduct is produced. Other suitable gelling agents includemethylhydroxycellulose, polyoxyethylene-polyoxypropylene,hydroxy-ethylcellulose and gelatin. Although gels commonly employaqueous carrier liquid, alcohols and oils can be used as the carrierliquid as well.

Various additives, known to those skilled in the art, may be included informulations, e.g., topical formulations. Examples of additives include,but are not limited to, solubilizers, skin permeation enhancers,opacifiers, preservatives (e.g., anti-oxidants), gelling agents,buffering agents, surfactants (particularly nonionic and amphotericsurfactants), emulsifiers, emollients, thickening agents, stabilizers,humectants, colorants, fragrance, and the like. Inclusion ofsolubilizers and/or skin permeation enhancers is particularly preferred,along with emulsifiers, emollients and preservatives. An optimum topicalformulation comprises approximately: 2 wt. % to 60 wt. %, preferably 2wt. % to 50 wt. %, solubilizer and/or skin permeation enhancer, 2 wt. %to 50 wt. %, preferably 2 wt. % to 20 wt. %, emulsifiers; 2 wt. % to 20wt. % emollient; and 0.01 to 0.2 wt. % preservative, with the activeagent and carrier (e.g., water) making of the remainder of theformulation.

A skin permeation enhancer serves to facilitate passage of therapeuticlevels of active agent to pass through a reasonably sized area ofunbroken skin. Suitable enhancers are well known in the art and include,for example: lower alkanols such as methanol ethanol and 2-propanol;alkyl methyl sulfoxides such as dimethylsulfoxide (DMSO),decylmethylsulfoxide (C.sub.10 MSO) and tetradecylmethyl sulfoxide;pyrrolidones such as 2-pyrrolidone, N-methyl-2-pyrrolidone andN-(-hydroxyethyl)pyrrolidone: urea; N,N-diethyl-m-toluamide;C.sub.2-C.sub.6 alkanediols; miscellaneous solvents such as dimethylformamide (DMF), N,N-dimethylacetamide (DMA) and tetrahydrofurfurylalcohol; and the 1-substituted azacycloheptan-2-ones, particularly1-n-dodecylcyclazacycloheptan-2-one (laurocapram; available under thetrademark Azone.sup.RTM from Whitby Research Incorporated, Richmond,Va.).

Examples of solubilizers include, but are not limited to, the following:hydrophilic ethers such as diethylene glycol monoethyl ether(ethoxydiglycol, available commercially as Transcutol.sup.RTM) anddiethylene glycol monoethyl ether oleate (available commercially asSoficutol.sup.RTM); polyethylene castor oil derivatives such as polyoxy35 castor oil, polyoxy 40 hydrogenated castor oil, etc.; polyethyleneglycol, particularly lower molecular weight polyethylene glycols such asPEG 300 and PEG 400, and polyethylene glycol derivatives such as PEG-8caprylic/capric glycerides (available commercially as Labrasol.sup.RTM);alkyl methyl sulfoxides such as DMSO; pyrrolidones such as 2-pyrrolidoneand N-methyl-2-pyrrolidone; and DMA. Many solubilizers can also act asabsorption enhancers. A single solubilizer may be incorporated into theformulation, or a mixture of solubilizers may be incorporated therein.

Suitable emulsifiers and co-emulsifiers include, without limitation,those emulsifiers and co-emulsifiers described with respect tomicroemulsion formulations. Emollients include, for example, propyleneglycol, glycerol, isopropyl myristate, polypropylene glycol-2 (PPG-2)myristyl ether propionate, and the like.

Other active agents may also be included in formulations, e.g., otheranti-inflammatory agents, analgesics, antimicrobial agents, antifungalagents, antibiotics, vitamins, antioxidants, and sunblock agentscommonly found in sunscreen formulations including, but not limited to,anthranilates, benzophenones (particularly benzophenone-3), camphorderivatives, cinnamates (e.g., octyl methoxycinnamate), dibenzoylmethanes (e.g., butyl methoxydibenzoyl methane), p-aminobenzoic acid(PABA) and derivatives thereof, and salicylates (e.g., octylsalicylate).

In one embodiment the oligonucleotides are specific for polynucleotidesof a Sirtuin (SIRT), which includes, without limitation noncodingregions. The Sirtuin (SIRT) targets comprise variants of a Sirtuin(SIRT); mutants of a Sirtuin (SIRT), including SNPs; noncoding sequencesof a Sirtuin (SIRT); alleles, fragments and the like. Preferably theoligonucleotide is an antisense RNA molecule.

In accordance with embodiments of the invention, the target nucleic acidmolecule is not limited to a Sirtuin (SIRT) polynucleotides alone butextends to any of the isoforms, receptors, homologs, non-coding regionsand the like of a Sirtuin (SIRT).

In another embodiment, an oligonucleotide targets a natural antisensesequence (natural antisense to the coding and non-coding regions) of aSirtuin (SIRT) targets, including, without limitation, variants,alleles, homologs, mutants, derivatives, fragments and complementarysequences thereto. Preferably the oligonucleotide is an antisense RNA orDNA molecule.

In another embodiment, the oligomeric compounds of the present inventionalso include variants in which a different base is present at one ormore of the nucleotide positions in the compound. For example, if thefirst nucleotide is an adenine, variants may be produced which containthymidine, guanosine, cytidine or other natural or unnatural nucleotidesat this position. This may be done at any of the positions of theantisense compound.

In some embodiments, homology, sequence identity or complementarity;between the antisense compound and target is from about 50% to about60%. In some embodiments, homology, sequence identity orcomplementarity, is from about 60% to about 70%. In some embodiments,homology, sequence identity or complementarity, is from about 70% toabout 80%. In some embodiments, homology, sequence identity orcomplementarity, is from about 80% to about 90%. In some embodiments,homology, sequence identity or complementarity, is about 90%, about 92%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100%.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired. Such conditionsinclude, i.e., physiological conditions in the case of in vivo assays ortherapeutic treatment, and conditions in which assays are performed inthe case of in vitro assays.

An antisense compound, whether DNA, RNA, chimeric, substituted etc, isspecifically hybridizable when binding of the compound to the target DNAor RNA molecule interferes with the normal function of the target DNA orRNA to cause a loss of utility, and there is a sufficient degree ofcomplementarily to avoid non-specific binding of the antisense compoundto non-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and in the case of in vitro assays,under conditions in which the assays are performed.

In another embodiment, targeting of a Sirtuin (SIRT) including withoutlimitation, antisense sequences which are identified and expanded, usingfor example, PCR, hybridization etc., one or more of the sequences setforth as SEQ ID NO: 5 to 14, and the like, modulate the expression orfunction of a Sirtuin (SIRT). In one embodiment, expression or functionis up-regulated as compared to a control. In another embodiment,expression or function is down-regulated as compared to a control.

In another embodiment, oligonucleotides comprise nucleic acid sequencesset forth as SEQ ID NOS: 15 to 94 including antisense sequences whichare identified and expanded, using for example, PCR, hybridization etc.These oligonucleotides can comprise one or more modified nucleotides,shorter or longer fragments, modified bonds and the like. Examples ofmodified bonds or internucleotide linkages comprise phosphorothioate,phosphorodithioate or the like. In another embodiment, the nucleotidescomprise a phosphorus derivative. The phosphorus derivative (or modifiedphosphate group) which may be attached to the sugar or sugar analogmoiety in the modified oligonucleotides of the present invention may bea monophosphate, diphosphate, triphosphate, alkylphosphate,alkanephosphate, phosphorothioate and the like. The preparation of theabove-noted phosphate analogs, and their incorporation into nucleotides,modified nucleotides and oligonucleotides, per se, is also known andneed not be described here.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotides have been safelyand effectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

In embodiments of the present invention oligomeric antisense compounds,particularly oligonucleotides, bind to target nucleic acid molecules andmodulate the expression and/or function of molecules encoded by a targetgene. The functions of DNA to be interfered comprise, for example,replication and transcription. The functions of RNA to be interferedcomprise all vital functions such as, for example, translocation of theRNA to the site of protein translation, translation of protein from theRNA, splicing of the RNA to yield one or more mRNA species, andcatalytic activity which may be engaged in or facilitated by the RNA.The functions may be up-regulated or inhibited depending on thefunctions desired.

The antisense compounds, include, antisense oligomeric compounds,antisense oligonucleotides, external guide sequence (EGS)oligonucleotides, alternate splicers, primers, probes, and otheroligomeric compounds that hybridize to at least a portion of the targetnucleic acid. As such, these compounds may be introduced in the form ofsingle-stranded, double-stranded, partially single-stranded, or circularoligomeric compounds.

Targeting an antisense compound to a particular nucleic acid molecule,in the context of this invention, can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose function is to be modulated. This target nucleic acid may be, forexample, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes a Sirtuin (SIRT).

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites” as used in the present invention, aredefined as positions within a target nucleic acid.

In one embodiment, the antisense oligonucleotides bind to the naturalantisense sequences of a Sirtuin (SIRT) and modulate the expressionand/or function of a Sirtuin (SIRT) (SEQ ID NO: 1 to 3). Examples ofantisense sequences include SEQ ID NOS: 4 to 29.

In another embodiment, the antisense oligonucleotides bind to one ormore segments of a Sirtuin (SIRT) polynucleotide and modulate theexpression and/or function of a Sirtuin (SIRT). The segments comprise atleast five consecutive nucleotides of a Sirtuin (SIRT) sense orantisense polynucleotides.

In another embodiment, the antisense oligonucleotides are specific fornatural antisense sequences of a Sirtuin (SIRT) Wherein binding of theoligonucleotides to the natural antisense sequences of a Sirtuin (SIRT)modulate expression and/or function of a Sirtuin (SIRT).

In another embodiment, oligonucleotide compounds comprise sequences setforth as SEQ ID NOS: 15 to 94, antisense sequences which are identifiedand expanded, using for example, PCR, hybridization etc Theseoligonucleotides can comprise one or more modified nucleotides, shorteror longer fragments, modified bonds and the like. Examples of modifiedbonds or internucleotide linkages comprise phosphorothioate,phosphorodithioate or the like. In another embodiment, the nucleotidescomprise a phosphorus derivative. The phosphorus derivative (or modifiedphosphate group) which may be attached to the sugar or sugar analogmoiety in the modified oligonucleotides of the present invention may bea monophosphate, diphosphate, triphosphate, alkylphosphate,alkanephosphate, phosphorothioate and the like. The preparation of theabove-noted phosphate analogs, and their incorporation into nucleotides,modified nucleotides and oligonucleotides, per se, is also known andneed not be described here.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules: 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes has a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG; and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes).Eukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA transcribed from a geneencoding a Sirtuin (SIRT), regardless of the sequence(s) of suchcottons. A translation termination codon (or “stop codon”) of a gene mayhave one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (thecorresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA,respectively).

The terms “start codon region” and “translation initiation cottonregion” refer to a portion of such an mRNA or gene that encompasses fromabout 25 to about 50 contiguous nucleotides in either direction (i.e.,5′ or 3′) from a translation initiation codon. Similarly, the terms“stop codon region” and “translation termination cotton region” refer toa portion of such an mRNA or gene that encompasses from about 25 toabout 50 contiguous nucleotides in either direction (i.e., 5′ or 3′)from a translation termination cotton. Consequently, the “start codonregion” (Or “translation initiation codon region”) and the “stop codonregion” (or “translation termination codon legion”) are all regions thatmay be targeted effectively with the antisense compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, atargeted region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Another target region includes the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene). Still another target legionincludes the 3′ untranslated region (3′UTR), known in the art to referto the portion of an mRNA in the 3′ direction from the translationtermination codon, and thus including nucleotides between thetranslation termination codon and 3′ end of an mRNA (or correspondingnucleotides on the gene). The 5′ cap site of an mRNA comprises anN7-methylated guanosine residue joined to the 5′-most residue of thetRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap site. Another target region for thisinvention is the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. In one embodiment, targeting splicesites, i.e., intron-exon junctions or exon-intron junctions, isparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular splice product isimplicated in disease. An aberrant fusion junction due to rearrangementor deletion is another embodiment of a target site. mRNA transcriptsproduced via the process of splicing of two (or more) mRNAs fromdifferent gene sources are known as “fusion transcripts”. Introns can beeffectively targeted using antisense compounds targeted to, for example,DNA or pre-mRNA.

In another embodiment, the antisense oligonucleotides bind to codingand/or non-coding regions of a target polynucleotide and modulate theexpression and/or function of the target molecule.

In another embodiment, the antisense oligonucleotides bind to naturalantisense polynucleotides and modulate the expression and/or function ofthe target molecule.

In another embodiment, the antisense oligonucleotides bind to sensepolynucleotides and modulate the expression and/or function of thetarget molecule.

Alternative RNA transcripts can be produced from the same genomic regionof DNA. These alternative transcripts are generally known as “variants”.More specifically, “pre-mRNA variants” are transcripts produced from thesame genomic DNA that differ from other transcripts produced from thesame genomic DNA in either their start or stop position and contain bothintronic and exonic sequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

Variants can be produced through the use of alternative signals to startor stop transcription. Pre-mRNAs and mRNAs can possess more than onestart codon or stop codon. Variants that originate from a pre-mRNA ormRNA that use alternative start codons are known as “alternative startvariants” of that pre-mRNA or mRNA. Those transcripts that use analternative stop codon are known as “alternative stop variants” of thatpre-mRNA or mRNA. One specific type of alternative stop variant is the“polyA variant” in which the multiple transcripts produced result fromthe alternative selection of one of the “polyA stop signals” by thetranscription machinery, thereby producing transcripts that terminate atunique polyA sites. Within the context of the invention, the types ofvariants described herein are also embodiments of target nucleic acids.

The locations on the target nucleic acid to which the antisensecompounds hybridize are defined as at least a 5-nucleotide long portionof a target region to Which an active antisense compound is targeted.

While the specific sequences of certain exemplary target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional target segments are readilyidentifiable by one having ordinary skill in the art in view of thisdisclosure.

Target segments 5-100 nucleotides in length comprising a stretch of atleast five (5) consecutive nucleotides selected from within theillustrative target segments are considered to be suitable for targetingas well.

Target segments can include DNA or RNA sequences that comprise at leastthe 5 consecutive nucleotides from the 5′-terminus of one of theillustrative target segments (the remaining nucleotides being aconsecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 5 to about 100 nucleotides). Similarlytarget segments are represented by DNA or RNA sequences that comprise atleast the 5 consecutive nucleotides from the 3′-terminus of one of theillustrative target segments (the remaining nucleotides being aconsecutive stretch of the same DNA or RNA beginning immediatelydownstream of the 3′-terminus of the target segment and continuing untilthe DNA or RNA contains about 5 to about 100 nucleotides). One havingskill in the art armed with the target segments illustrated herein willbe able, without undue experimentation, to identify further targetsegments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

In embodiments of the invention the oligonucleotides bind to anantisense strand of a particular target. The oligonucleotides are atleast 5 nucleotides in length and can be synthesized so eacholigonucleotide targets overlapping sequences such that oligonucleotidesare synthesized to cover the entire length of the target polynucleotide.The targets also include coding as well as non coding regions.

In one embodiment, specific nucleic acids are targeted by antisenseoligonucleotides. Targeting an antisense compound to a particularnucleic acid, is a multistep process. The process usually begins withthe identification of a nucleic acid sequence whose function is to bemodulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or anon coding polynucleotide suchas for example, non coding RNA (ncRNA).

RNAs can be classified into (1) messenger RNAs (mRNAs), which aretranslated into proteins, and (2) non-protein-coding RNAs (ncRNAs).ncRNAs comprise microRNAs, antisense transcripts and otherTranscriptional Units (TU) containing a high density of stop codons andlacking any extensive “Open Reading Frame”. Many ncRNAs appear to startfrom initiation sites in 3′ untranslated regions (3′UTRs) ofprotein-coding loci. ncRNAs are often rare and at least half of thencRNAs that have been sequenced by the FANTOM consortium seem not to bepolyadenylated. Most researchers have for obvious reasons focused onpolyadenylated mRNAs that are processed and exported to the cytoplasm.Recently, it was shown that the set of non-polyadenylated nuclear RNAsmay be very lame, and that many such transcripts arise from intergenicregions. The mechanism by which ncRNAs may regulate gene expression isby base pairing with target transcripts. The RNAs that function by basepairing can be grouped into (1) cis encoded RNAs that are encoded at thesame genetic location, but on the opposite strand to the RNAs they actupon and therefore display perfect complementarity to their target, and(2) trans-encoded RNAs that are encoded at a chromosomal locationdistinct from the RNAs they act upon and generally do not exhibitperfect base-pairing potential with their targets.

Without wishing to be bound by theory, perturbation of an antisensepolynucleotide by the antisense oligonucleotides described herein canalter the expression of the corresponding sense messenger RNAs. However,this regulation can either be discordant (antisense knockdown results inmessenger RNA elevation) or concordant (antisense knockdown results inconcomitant messenger RNA reduction). In these cases, antisenseoligonucleotides can be targeted to overlapping or non-overlapping partsof the antisense transcript resulting in its knockdown or sequestration.Coding as well as non-coding antisense can be targeted in an identicalmanner and that either category is capable of regulating thecorresponding sense transcripts—either in a concordant or disconcordantmanner. The strategies that are employed in identifying newoligonucleotides for use against a target can be based on the knockdownof antisense RNA transcripts by antisense oligonucleotides or any othermeans of modulating the desired target.

Strategy 1: In the case of discordant regulation, knocking down theantisense transcript elevates the expression of the conventional (sense)gene. Should that latter gene encode for a known or putative drugtarget, then knockdown of its antisense counterpart could conceivablymimic the action of a receptor agonist or an enzyme stimulant.

Strategy 2: In the case of concordant regulation, one couldconcomitantly knock down both antisense and sense transcripts andthereby achieve synergistic reduction of the conventional (sense) geneexpression. If for example, an antisense oligonucleotide is used toachieve knockdown, then this strategy can be used to apply one antisenseoligonucleotide targeted to the sense transcript and another antisenseoligonucleotide to the corresponding antisense transcript, or a singleenergetically symmetric antisense oligonucleotide that simultaneouslytargets overlapping sense and antisense transcripts.

According to the present invention, antisense compounds includeantisense oligonucleotides, ribozymes, external guide sequence (EGS)oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, and otheroligomeric compounds which hybridize to at (east a portion of the targetnucleic acid and modulate its function. As such, they may be DNA, RNA,DNA-like, RNA-like, or mixtures thereof, or may be mimetics of one ormore of these. These compounds may be single-stranded, doublestranded,circular or hairpin oligomeric compounds and may contain structuralelements such as internal or terminal bulges, mismatches or loops.Antisense compounds are routinely prepared linearly but can be joined orotherwise prepared to be circular and/or branched. Antisense compoundscan include constructs such as, for example, two strands hybridized toform a wholly or partially double-stranded compound or a single strandwith sufficient self-complementarity to allow for hybridization andformation of a fully or partially double-stranded compound. The twostrands can be linked internally leaving free 3′ or 5′ termini or can belinked to form a continuous hairpin structure or loop. The hairpinstructure may contain an overhang on either the 5′ or 3′ terminusproducing an extension of single stranded character. The double strandedcompounds optionally can include overhangs on the ends. Furthermodifications can include conjugate groups attached to one of thetermini, selected nucleotide positions, sugar positions or to one ofinternucleoside linkages. Alternatively, the two strands can be linkedvia a non-nucleic acid moiety or linker group. When formed from only onestrand, dsRNA can take the form of a self-complementary hairpin-typemolecule that doubles back on itself to form a duplex. Thus, the dsRNAscan be fully or partially double stranded. Specific modulation of geneexpression can be achieved by stable expression of dsRNA hairpins intransgenic cell lines, however, in some embodiments, the gene expressionor function is up regulated. When formed from two strands, or a singlestrand that takes the farm of a self-complementary hairpin-type moleculedoubled back on itself to form a duplex, the two strands (orduplex-forming regions of a single strand) are complementary RNA strandsthat base pair in Watson-Crick fashion.

Once introduced to a system, the compounds of the invention may elicitthe action of one or more enzymes or structural proteins to effectcleavage or other modification of the target nucleic acid or may workvia occupancy-based mechanisms. In general, nucleic acids (includingoligonucleotides) may be described as “DNA-like” (i.e., generally havingone or more 2′-deoxy sugars and, generally, T rather than U bases) or“RNA-like” (i.e., generally having one or more 2′-hydroxyl or2′-modified sugars and, generally U rather than T bases). Nucleic acidhelices can adopt more than one type of structure, most commonly the A-and B-forms. It is believed that, in general, oligonucleotides whichhave B-form-like structure are “DNA-like” and those which haveA-formlike structure are “RNA-like.” In some (chimeric) embodiments, anantisense compound may contain both A- and B-form regions.

In another embodiment, the desired oligonucleotides or antisensecompounds, comprise at least one of: antisense RNA, antisense DNA,chimeric antisense oligonucleotides, antisense oligonucleotidescomprising modified linkages, interference RNA (RNAi), short interferingRNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA(stRNA); or a short, hairpin RNA (shRNA); small RNA-induced geneactivation (RNAa); small activating RNAs (saRNAs), or combinationsthereof.

dsRNA can also activate gene expression, a mechanism that has beentermed “small RNA-induced gene activation” or RNAa. dsRNAs targetinggene prompters induce potent transcriptional activation of associatedgenes. RNAa was demonstrated in human cells using synthetic dsRNAs,termed “small activating RNAs” (saRNAs).

Small double-stranded RNA (dsRNA), such as small interfering RNA (siRNA)and microRNA (miRNA), have been found to be the trigger of anevolutionary conserved mechanism known as RNA interference (RNAi). RNAiinvariably leads to gene silencing. However, in instances described indetail in the examples section which follows, oligonucleotides are shownto increase the expression and/or function of the Sirtuin (SIRT)polynucleotides and encoded products thereof dsRNAs may also act assmall activating RNAs (saRNA). Without wishing to be bound by theory, bytargeting sequences in gene promoters, saRNAs would induce target geneexpression in a phenomenon referred to as dsRNA-induced transcriptionalactivation (RNAa).

In a further embodiment, the “target segments” identified herein may beemployed in a screen for additional compounds that modulate theexpression of a Sirtuin (SIRT) polynucleotide. “Modulators” are thosecompounds that decrease or increase the expression of a nucleic acidmolecule encoding a Sirtuin (SIRT) and which comprise at least a5-nucleotide portion that is complementary to a target segment. Thescreening method comprises the steps of contacting a target segment of anucleic acid molecule encoding sense or natural antisensepolynucleotides of a Sirtuin (SIRT) with one or more candidatemodulators, and selecting for one or more candidate modulators whichdecrease or increase the expression of a nucleic acid molecule encodinga Sirtuin (SIRT) polynucleotide, e.g. SEQ ID NOS: 15 to 94. Once it isshown that the candidate modulator or modulators are capable ofmodulating (e.g. either decreasing or increasing) the expression of anucleic acid molecule encoding a Sirtuin (SIRT) polynucleotide, themodulator may then be employed in further investigative studies of thefunction of a Sirtuin (SIRT) polynucleotide, or for use as a research,diagnostic, or therapeutic agent in accordance with the presentinvention.

Targeting the natural antisense sequence modulates the function of thetarget gene. For example, the Sirtuin (SIRf) (e.g. accession numbersNM_(—)012238.3, NM_(—)001159589, NM_(—)012239, NM_(—)16539), in aembodiment, the target is an antisense polynucleotide of the Sirtuin(SIRT). In a embodiment, an antisense oligonucleotide targets senseand/or natural antisense sequences of a Sirtuin (SIRT) polynucleotide(e.g. accession numbers NM₁₃ 0122383, NM_(—)001159589, NM_(—)012239,NM_(—)416539), variants, alleles, isoforms, homologs, mutants,derivatives, fragments and complementary sequences thereto. Preferablythe oligonucleotide is an antisense molecule and the targets includecoding and noncoding regions of antisense and/or sense Sirtuin (SIRT)polynucleotides.

The target segments of the present invention may be also be combinedwith their respective complementary antisense compounds of the presentinvention to form stabilized double-stranded (duplexed)oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocessing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications. For example, suchdouble-stranded moieties have been shown to inhibit the target by theclassical hybridization of antisense strand of the duplex to the target,thereby triggering enzymatic degradation of the target.

In a embodiment, an antisense oligonucleotide targets Sirtuin (SIRT)polynucleotides (e.g. accession numbers NM_(—)0122383, NM_(—)001159589,NM_(—)012239, NM_(—)016539), variants, alleles, isoforms, homologs,mutants, derivatives, fragments and complementary sequences thereto.Preferably the oligonucleotide is an antisense molecule.

In accordance with embodiments of the invention, the target nucleic acidmolecule is not limited to Sirtuin (SIRT) alone but extends to any ofthe isoforms, receptors, homologs and the like of a Sirtuin (SIRE)molecule.

In another embodiment, an oligonucleotide targets a natural antisensesequence of a Sirtuin (SIRT) polynucleotide, for example,polynucleotides set forth as SEQ ID NO: 5 to 14, and any variants,alleles, homologs, mutants, derivatives, fragments and complementarysequences thereto. Examples of antisense oligonucleotides are set forthas SEQ ID NOS:15 to 94.

In one embodiment, the oligonucleotides are complementary to or bind tonucleic acid sequences of a Sirtuin (SIRT) antisense, including withoutlimitation noncoding sense and/or antisense sequences associated with aSirtuin (SIRT) polynucleotide and modulate expression and/or function ofa Sirtuin (SIRT) molecule.

In another embodiment, the oligonucleotides are complementary to or bindto nucleic acid sequences of a Sirtuin (SIRT) natural antisense, setforth as SEQ ID NO: 5 to 14 and modulate expression and/or function of aSirtuin (SIRT) molecule.

In a embodiment, oligonucleotides comprise sequences of at least 5consecutive nucleotides of SEQ ID NOS: 15 to 94 and modulate expressionand/or function of a Sirtuin (SIRT) molecule.

The polynucleotide targets comprise Sirtuin (SIRT), including familymembers thereof, variants of a Sirtuin (SIRT); mutants of a Sirtuin(SIRT), including SNPs; noncoding sequences of a Sirtuin (SIRT): allelesof a Sirtuin (SIRT); species variants, fragments and the like.Preferably the oligonucleotide is an antisense molecule.

In another embodiment, the oligonucleotide targeting Sirtuin (SIRT)polynucleotides, comprise: antisense RNA, interference RNA (RNAi), shortinterfering RNA (siRNA); micro interfering RNA (miRNA); a small,temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-inducedgene activation (RNAa); or, small activating RNA (saRNA).

In another embodiment, targeting of a Sirtuin (SIRT) polynucleotide,e.g. SEQ ID NO: 5 to 14 modulate the expression or function of thesetargets. In one embodiment, expression or function is up-regulated ascompared to a control. In another embodiment, expression or function isdown-regulated as compared to a control.

In another embodiment, antisense compounds comprise sequences set forthas SEQ ID NOS: 15 to 94. These oligonucleotides can comprise one or moremodified nucleotides, shorter or longer fragments, modified bonds andthe like.

In another embodiment, SEQ ID NOS: 0.15 to 94 comprise one or more LNAnucleotides.

The modulation of a desired target nucleic acid can be carried out inseveral ways known in the art. For example, antisense oligonucleotides,siRNA etc. Enzymatic nucleic acid molecules (e.g., ribozymes) arenucleic acid molecules capable of catalyzing one or more of a variety ofreactions, including the ability to repeatedly cleave other separatenucleic acid molecules in a nucleotide base sequence-specific manner.Such enzymatic nucleic acid molecules can be used, for example, totarget virtually any RNA transcript.

Because of their sequence-specificity, trans-cleaving enzymatic nucleicacid molecules show promise as therapeutic agents for human disease.Enzymatic nucleic acid molecules can be designed to cleave specific RNAtargets within the background of cellular RNA. Such a cleavage eventrenders the mRNA non-functional and abrogates protein expression fromthat RNA. In this manner, synthesis of a protein associated with adisease state can be selectively inhibited.

In general, enzymatic nucleic acids with RNA cleaving activity act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of a enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

Several approaches such as in vitro selection (evolution) strategieshave been used to evolve new nucleic acid catalysts capable ofcatalyzing a variety of reactions, such as cleavage and ligation ofphosphodiester linkages and amide linkages.

The development of ribozymes that are optimal for catalytic activitywould contribute significantly to any strategy that employs RNA-cleavingribozymes for the purpose of regulating gene expression. The hammerheadribozyme, for example, functions with a catalytic rate (kcat) of about 1min-1 in the presence of saturating (10 mM) concentrations of Mg2+cofactor. An artificial “.RNA ligase” ribozytne has been shown tocatalyze the corresponding self-modification reaction with a rate ofabout 100 min-1. In addition, it is known that certain modifiedhammerhead ribozymes that have substrate binding arms made of DNAcatalyze RNA cleavage with multiple rum-over rates that approach 100min-1. Finally, replacement of a specific residue within the catalyticcore of the hammerhead with certain nucleotide analogues gives modifiedribozymes that show as much as a 10-fold improvement in catalytic rate.These findings demonstrate that ribozymes can promote chemicaltransformations with catalytic rates that are significantly greater thanthose displayed in vitro by most natural self-cleaving ribozymes. It isthen possible that the structures of certain selibleaving ribozymes maybe optimized to give maximal catalytic activity, or that entirely newRNA motifs can be made that display significantly faster rates for RNAphosphodiester cleavage.

Intermolecular cleavage of an RNA substrate by an RNA catalyst that fitsthe “hammerhead” model was first shown in 1987. The RNA catalyst wasrecovered and reacted with multiple RNA molecules, demonstrating that itwas truly catalytic.

Catalytic RNAs designed based on the “hammerhead” motif have been usedto cleave specific target sequences by making appropriate base changesin the catalytic RNA to maintain necessary base pairing with the targetsequences. This has allowed use of the catalytic RNA to cleave specifictarget sequences and indicates that catalytic RNAs designed according tothe “hammerhead” model may possibly cleave specific substrate RNAs invivo.

RNA interference (RNAi) has become a powerful tool for modulating geneexpression in mammals and mammalian cells. This approach requires thedelivery of small interfering RNA (siRNA) either as RNA itself or asDNA, using an expression plasmid or virus and the coding sequence forsmall hairpin RNAs that are processed to siRNAs. This system enablesefficient transport of the pre-siRNAs to the cytoplasm where they areactive and permit the use of regulated and tissue specific promoters forgene expression.

In one embodiment, an oligonucleotide or antisense compound comprises anoligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleicacid (DNA), or a mimetic, chimera, analog or homolog thereof. This termincludes oligonucleotides composed of naturally occurring nucleotides,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftendesired over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a targetnucleic acid and increased stability in the presence of nucleases.

According to the present invention, the oligonucleotide or “antisensecompounds” include antisense oligonucleotides (e.g. RNA, DNA, mimetic,chimera, analog or homolog thereof), ribozymes, external guide sequence(EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, snRNA, aRNA, andother oligomeric compounds which hybridize to at least a portion of thetarget nucleic acid and modulate its function. As such they may be DNA,RNA, DNA-like, RNA-like, or mixtures thereof, or may be mimetics of oneor more of these. These compounds may be single-stranded,double-stranded, circular or hairpin oligomeric compounds and maycontain structural elements such as internal or terminal bulges,mismatches or loops. Antisense compounds are routinely prepared linearlybut can be joined or otherwise prepared to be circular and/or branched.Antisense compounds can include constructs such as, for example, twostrands hybridized to form a wholly or partially double-strandedcompound or a single strand with sufficient self-complementarity toallow for hybridization and formation of a fully or partiallydouble-stranded compound. The two strands can be linked internallyleaving free 3′ or 5′ termini or can be linked to form a continuoushairpin structure or loop. The hairpin structure may contain an overhangon either the 5′ or 3′ terminus producing an extension of singlestranded character. The double stranded compounds optionally can includeoverhangs on the ends. Further modifications can include conjugategroups attached to one of the termini, selected nucleotide positions,sugar positions or to one of the internucleoside linkages.Alternatively, the two strands can be linked via anon-nucleic acidmoiety or linker group. When formed from only one strand, dsRNA can takethe form of a self-complementary hairpin-type molecule that doubles backon itself to form a duplex. Thus, the dsRNAs can be fully or partiallydouble stranded. Specific modulation of gene expression can be achievedby stable expression of dsRNA hairpins in transgenic cell lines. Whenformed from two strands, or a single strand that takes the form of aself-complementary hairpin-type molecule doubled back on itself to forma duplex, the two strands (or duplex-forming regions of a single strand)are complementary RNA strands that base pair in Watson-Crick fashion,

Once introduced to a system, the compounds of the invention may elicitthe action of one or more enzymes or structural proteins to effectcleavage or other modification of the target nucleic acid or may workvia occupancy-based mechanisms. In general, nucleic acids (includingoligonucleotides) may be described as “DNA-like” (i.e., generally havingone or more 2′-deoxy sugars and, generally, T rather than U bases) or“RNA-like” (i.e., generally having one or more 2′-hydroxyl or2′-modified sugars and generally U rather than 1 bases). Nucleic acidhelices can adopt more than one type of structure, most commonly the A-and B-forms. It is believed that, in general, oligonucleotides whichhave B-form-like structure are “DNA-like” and those which haveA-formlike structure are “RNA-like.” In some (chimeric) embodiments, anantisense compound may contain both A- and B-form regions.

The antisense compounds in accordance with this invention can comprisean antisense portion from about 5 to about 80 nucleotides (.i.e. fromabout 5 to about 80 linked nucleosides) in length. This refers to thelength of the antisense strand or portion of the antisense compound. Inother words, a single-stranded antisense compound of the inventioncomprises from 5 to about 80 nucleotides, and a double-strandedantisense compound of the invention (such as a dsRNA, for example)comprises a sense and an antisense strand or portion of 5 to about 80nucleotides in length. One of ordinary skill in the art will appreciatethat this comprehends antisense portions of 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides inlength, or any range therewithin.

In one embodiment, the antisense compounds of the invention haveantisense portions of 10 to 50 nucleotides in length. One havingordinary skill in the art will appreciate that this embodiesoligonucleotides having antisense portions of 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleotides in length, or any range therewithin. In some embodiments,the oligonucleotides are 15 nucleotides in length.

In one embodiment, the antisense or oligonucleotide compounds of theinvention have antisense portions of 12 or 13 to 30 nucleotides inlength. One having ordinary skill in the art will appreciate that thisembodies antisense compounds having antisense portions of 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nucleotides in length, or any range therewithin.

In another embodiment, the oligomeric compounds of the present inventionalso include variants in which a different base is present at one ormore of the nucleotide positions in the compound. For example, if thefirst nucleotide is an adenosine, variants may be produced which containthymidine, guanosine or cytidine at this position. This may be done atany of the positions of the antisense or dsRNA compounds. Thesecompounds are then tested using the methods described herein todetermine their ability to inhibit expression of a target nucleic acid.

In some embodiments, homology, sequence identity or complementarity,between the antisense compound and target is from about 40% to about60%. In some embodiments, homology, sequence identity orcomplementarity, is from about 60% to about 70%. In some embodiments,homology; sequence identity or complementarity, is from about 70% toabout 80%. In some embodiments, homology; sequence identity orcomplementarity, is from about 80% to about 90%. In some embodiments,homology, sequence identity or complementarity, is about 90%, about 92%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100%.

In another embodiment, the antisense oligonucleotides, such as forexample, nucleic acid molecules set forth in SEQ ID NOS: 4 to 29comprise one or more substitutions or modifications. In one embodiment,the nucleotides are substituted with locked nucleic acids (LNA).

In another embodiment, the oligonucleotides target one or more regionsof the nucleic acid molecules sense and/or antisense of coding and/ornon-coding sequences associated with Sirtuin (SIRT) and the sequencesset forth as SEQ ID NOS: 1 to 14. The oligonucleotides are also targetedto overlapping regions of SEQ ID NOS: 1 to 14.

Certain oligonucleotides of this invention are chimericoligonucleotides. “Chimeric oligonucleotides” or “chimeras,” in thecontext of this invention, are oligonucleotides which contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionof modified nucleotides that confers one or more beneficial properties(such as, for example, increased nuclease resistance, increased uptakeinto cells, increased binding affinity for the target) and a region thatis a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of antisense modulation of gene expression.Consequently, comparable results can often be obtained with shorteroligonucleotides when chimeric oligonucleotides are used, compared tophosphorothioate deoxyoligonucleotides hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art. In one embodiment, a chimericoligonucleotide comprises at least one region modified to increasetarget binding affinity, and, usually, a region that acts as a substratefor RNAse H. Affinity of an oligonucleotide for its target (in thiscase, a nucleic acid encoding ras) is routinely determined by measuringthe Tm of an oligonucleotide/target pair, which is the temperature atwhich the oligonucleotide and target dissociate; dissociation isdetected spectrophotometrically. The higher the Tm, the greater is theaffinity of the oligonucleotide for the target.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotides mimetics as described above.Such; compounds have also been referred to in the an as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures comprise, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350, 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference.

In another embodiment, the region of the oligonucleotide which ismodified comprises at least one nucleotide modified at the 2′ positionof the sugar, most preferably a 2′-Oalkyl, 2′-O-alkyl-O-alkyl or2′-fluoro-modified nucleotide. In other embodiments, RNA modificationsinclude 2′-fluoro, 2′-amino and 2′ O-methyl modifications on the riboseof pyrimidines, abasic residues or an inverted base at the 3′ end of theRNA. Such modifications are routinely incorporated into oligonucleotidesand these oligonucleotides have been shown to have a higher Tm (i.e.,higher target binding affinity) than; 2′-deoxyoligonucleotides against agiven target The effect of such increased affinity is to greatly enhanceRNAi oligonucleotide inhibition of gene expression. RNAse H is acellular endonuclease that cleaves the RNA strand of RNA:DNA duplexes;activation of this enzyme therefore results in cleavage of the RNAtarget, and thus can greatly enhance the efficiency of RNAi inhibition.Cleavage of the RNA target can be routinely demonstrated by gelelectrophoresis. In another embodiment, the chimeric oligonucleotide isalso modified to enhance nuclease resistance. Cells contain a variety ofexo- and endo-nucleases which can degrade nucleic acids. A number ofnucleotide and nucleoside modifications have been shown to make theoligonucleotide into which they are incorporated more resistant tonuclease digestion than the native oligodeoxynucleotide. Nucleaseresistance is routinely measured by incubating oligonucleotides withcellular extracts or isolated nuclease solutions and measuring theextent of intact oligonucleotide remaining over time, usually by gelelectrophoresis. Oligonucleotides which have been modified to enhancetheir nuclease resistance survive intact for a longer time thanunmodified oligonucleotides. A variety of oligonucleotide modificationshave been demonstrated to enhance or confer nuclease resistance.Oligonucleotides which contain at least one phosphorothioatemodification are presently more preferred. In some cases,oligonucleotide modifications which enhance target binding affinity arealso, independently, able to enhance nuclease resistance. Some desirablemodifications can be found in De Mesmaeker et al. (1995) Acc. Chem.Res., 28:366-374.

Specific examples of some oligonucleotides envisioned for this inventioninclude those comprising modified backbones, for example,phosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most are oligonucleotides withphosphorothioate backbones and those with heteroatom backbones,particularly CH2-NH—O—CH2, CH, —N(CH3)-O—CH2 (known as amethylene(methylimino) or MMI backbone), CH2-O—N(CH3)-CH2,CH2-N(CH3)-N(CH3)-CH2 and O—N(CH3)-CH2-CH2 backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH). The amide baCkbonesdisclosed by De Mesmaeker et al. (1995) Acc. Chem. Res. 28:366-374 arealso preferred. Also are oligonucleotides having morpholino backbonestructures (Summerton and Weller, U.S. Pat. No. 5,034,506). In otherembodiments, such as the peptide nucleic acid (PNA) backbone, thephosphodiester backbone of the oligonucleotide is replaced with apolyamide backbone, the nucleotides being bound directly or indirectlyto the or) nitrogen atoms of the polyamide backbone. Oligonucleotidesmay also comprise one or more substituted sugar moietiesoligonucleotides comprise one of the following at the 2′ position: OH,SH, SCH3, F, OCN, OCH3, OCH3 O(CH2)n CH3, O(CH2)n NH2 or O(CH2)n CH3where n is from 1 to about 10; C1 to C10C lower alkyl, alkoxyalkoxy,substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O—,S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3;NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino;polyalkylamino; substituted silyl; an RNA cleaving group; a reportergroup; an intercalator; a group for improving the pharmacokineticproperties of an oligonucleotide; or a group for improving thepharmacodynamic properties of an oligonucleotide and other substituentshaving similar properties. A modification includes 2′-methoxyethoxy.[2-O—CH2 CH2 OCH3, also known as 2′-O-(2-methoxyethyl)]. Othermodifications include 2′-methoxy(2′-O—CH3), 2′-propoxy(2′-OCH2 CH2CH3)and 2′-fluoro (2′-F). Similar modifications may also be made at otherpositions on the oligonucleotide, particularly the 3′ position of thesugar on the 3′ terminal nucleotide and the 5′ position of 5′ terminalnucleotide. Oligonucleotides may also have sugar mimetics such ascyclobutyls in place of the pentofuranosyl group.

Oligonucleotides may also include, additionally or alternatively,nucleobase (often referred to in the art simply as “base”) modificationsor substitutions. As used herein, “unmodified” or “natural” nucleotidesinclude adenine (A), guanine (G), thymine (T), cytosine (C) and uracil(U). Modified nucleotides include nucleotides found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2′ deoxycytosine and often referred to in theart as 5-Me—C), 5-hydroxymethylcytosine (HMC), glucosyl HMC andgentobiosyl HMC, as well as synthetic nucleotides, e.g., 2-aminoadenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and2,6-diaminopurine. A “universal” base known in the art, e.g., inosine,may be included. 5-Me-C substitutions have been shown to increasenucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, V. S., in Crooke,S. T. and Lebleu, B., eds., Antisense Research and Applications, CRCPress, Boca Raton, 1993, pp. 276-278) and are presently basesubstitutions.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety; a cholesteryl moiety, athioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid. Oligonucleotidescomprising lipophilic moieties, and methods for preparing sucholigonucleotides are known in the art, for example, U.S. Pat. Nos.5,138,045, 5,218,105 and 5,459,255.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even atwithin a single nucleoside within an oligonucleotide. The presentinvention also includes oligonucleotides which are chimericoligonucleotides as hereinbefore defined.

In another embodiment, the nucleic acid molecule of the presentinvention is conjugated with another moiety including but not limited toabasic nucleotides, polyether, polyamine, polyamides, peptides,carbohydrates, lipid, or polyhydrocarbon compounds. Those skilled in theart will recognize that these molecules can be linked to one or more ofany nucleotides comprising the nucleic acid molecule at severalpositions on the sugar, base or phosphate group.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of one of ordinary skill in the art. It is alsowell known to use similar techniques to prepare other oligonucleotidessuch as the phosphorothioates and alkylated derivatives. It is also wellknown to use similar techniques and commercially available modifiedamidites and controlled-pore glass (CPG) products such as biotin,fluorescein, acridine or psoralen-modified amidites and/or CPG(available from Glen Research, Sterling Va.) to synthesize fluorescentlylabeled, biotinylated or other modified oligonucleotides such ascholesterol-modified oligonucleotides.

In accordance with the invention, use of modifications such as the useof LNA monomers to enhance the potency, specificity and duration ofaction and broaden the routes of administration of oligonucleotidescomprised of current chemistries such as MOE, ANA, FANA, PS etc. Thiscan be achieved by substituting some of the monomers in the currentoligonucleotides by LNA monomers. The LNA modified oligonucleotide mayhave a size similar to the parent compound or may be larger orpreferably smaller. It is that such LNA-modified oligonucleotidescontain less than about 70%, more preferably less than about 60%, mostpreferably less than about 50% LNA monomers and that their sizes arebetween about 5 and 25 nucleotides, more preferably between about 12 and20 nucleotides.

Modified oligonucleotide backbones comprise, but are not limited to,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates comprising 3′alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates comprising 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus containing linkages comprise, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides comprise, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5, 264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In other oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to azo nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds comprise, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5219,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen, et al. (1991) Science 254.1497-1500.

In another embodiment of the invention the oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular—CH2-NH—O—CH2-, —CH2-N(CH3)-O—CH2-known as amethylene (methylimino) or MMI backbone, —CH2-O—N(CH3)-CH2-,—CH2N(CH3)-N(CH3) CH2- and —O—N(CH3)-CH2-CH2- wherein the nativephosphodiester backbone is represented as —O—P—O—CH2- of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereference U.S. Pat. No. 5,602,240. Also are oligonucleotides havingmorpholino backbone structures of the above-referenced U.S. Pat. No.5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties oligonucleotides comprise one of the following at the 2′position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O-, S- orN-alkynyl; or O alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C to CO alkyl or C2 to CO alkenyland alkynyl. Particularly are O (CH2)n OmCH3, O(CH2)n, OCH3, O(CH2)nNH2,O(CH2)nCH3, O(CH2)nONH2, and O(CH2nON(CH2)nCH3)2 where n and m can befrom 1 to about 10. Other oligonucleotides comprise one of the followingat the 2′ position: C to CO, (lower alkyl, substitute lower alkyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN,CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oliganucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Amodification comprises 2′-methoxyethoxy(2′-O—CH2OCH3, also known as2′-O-(2-methoxyethyl) or 2′-MOE) i.e., an alkoxyalkoxy group. A furthermodification comprises 2′-dimethylaminooxyethoxy, i.e., aO(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′, -dimethylaminoethoxyethoxy (also known in the artas 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), 2′-O-CH2-O—CH2-N(CH2)2.

Other modifications comprise 2′-methoxy(2′-O CH3), 2′-aminopropoxy (2′-OCH2CH2CH2NR2) and 2′-fluoro (2′-F). Similar modifications may also bemade at other positions on the oligonucleotide, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures comprise, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and5,700,920, each of which is herein incorporated by reference.

Oligonucleotides may also comprise nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleotides comprise the purine bases adenine(A) and guanine (O), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleotides comprise other synthetic andnatural nucleotides such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosine,7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further, nucleotides comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopedia of PolymerScience And Engineering’, pages 858-859, Kroschwitz, J. I., ed. John.Wiley & Sons, 1990, those disclosed by Englisch et at, ‘AngewandleChemic, International Edition’, 1991, 30, page 613, and those disclosedby Sanghvi, Y. S., Chapter 15, ‘Antisense Research and Applications’,pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993.Certain of these nucleotides are particularly useful for increasing thebinding affinity of the oligomeric compounds of the invention. Thesecomprise 5-substituted pyrimidines, 6-azapyrimidines and N2, N-6 and 0-6substituted purines, comprising 2-aminopropyladenine, 5-propynyluraciland 5-propynyleytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds, ‘Antisense Research andApplications’, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently base substitutions, even more particularly when combined with2′-Omethoxyethyl sugar modifications.

Representative United States patents that teach the preparation of theabove noted modified nucleotides as well as other modified nucleotidescomprise, but are not limited to, U.S. Pat. Nos. 3,687,808, as well as4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941, each of whichis herein incorporated by reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates, which enhance the activity, cellular distribution, orcellular uptake of the oligonucleotide.

Such moieties comprise but are not limited to, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol, or undecylresidues, a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate apolyamine or a polyethylene glycol chain, or adarnantane acetic acid, apalmityl moiety, or an octadecylamine or hexylamino-carbonyl-toxycholesterol moiety.

Representative United States patents that teach the preparation of sucholigonucleotides conjugates comprise, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

Drug Discovery:

The compounds of the present invention can also be applied in the areasof drug discovery and target validation. The present inventioncomprehends the use of the compounds and target segments identifiedherein in drug discovery efforts to elucidate relationships that existbetween a Sirtuin (SIRT) polynucleotide and a disease state, phenotype,or condition. These methods include detecting or modulating a Sirtuin(SIRT) polynucleotide comprising contacting a sample, tissue, cell, ororganism with the compounds of the present invention, measuring thenucleic acid or protein level of a Sirtuin (SIRT) polynucleotide and/ora related phenotypic or chemical endpoint at some time after treatment,and optionally comparing the measured value to a non-treated sample orsample treated with a further compound of the invention. These methodscan also be performed in parallel or in combination with otherexperiments to determine the function of unknown genes for the processof target validation or to determine the validity of a particular geneproduct as a target for treatment or prevention of a particular disease,condition, or phenotype.

Assessing Up-Regulation or Inhibition of Gene Expression:

Transfer of an exogenous nucleic acid into a host cell or organism canbe assessed by directly detecting the presence of the nucleic acid inthe cell or organism. Such detection can be achieved by several methodswell known in the art. For example, the presence of the exogenousnucleic acid can be detected by Southern blot or by a polymerase chainreaction (PCR) technique using primers that specifically amplifynucleotide sequences associated with the nucleic acid. Expression of theexogenous nucleic acids can also be measured using conventional methodsincluding gene expression analysis. For instance, mRNA produced from anexogenous nucleic acid can be detected and quantified using a Northernblot and reverse transcription PCR (RT-PCR).

Expression of RNA from the exogenous nucleic acid can also be detectedby measuring an enzymatic activity or a reporter protein activity. Forexample, antisense modulatory activity can be measured indirectly as adecrease or increase in target nucleic acid expression as an indicationthat the exogenous nucleic acid is producing the effector RNA. Based onsequence conservation, primers can be designed and used to amplifycoding regions of the target genes. Initially, the most highly expressedcoding region from each gene can be used to build a model control gene,although any coding or non coding region can be used. Each control geneis assembled by inserting each coding region between a reporter codingregion and its poly(A) signal. These plasmids would produce an mRNA witha reporter gene in the upstream portion of the gene and a potential RNAitarget in the 3′ non-coding region. The effectiveness of individualantisense oligonucleotides would be assayed by modulation of thereporter gene. Reporter genes useful in the methods of the presentinvention include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP),cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase(Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivativesthereof. Multiple selectable markers are available that conferresistance to ampicillin, bleomycin, chloramphenicol, gentamycin,hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin,puromycin, and tetracycline. Methods to determine modulation of areporter gene are well known in the art, and include, but are notlimited to, fluorometric methods (e.g. fluorescence spectroscopy,Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy),antibiotic resistance determination.

SIRT1, SIRT3 and SIRT6 proteins and mRNA expression can be assayed usingmethods known to those of skill in the art and described elsewhereherein. For example, immunoassays such as the ELISA can be used tomeasure protein levels. Sirtuin (SIRT) antibodies for ELISAs areavailable commercially, e.g., from R&D Systems (Minneapolis, Minn.),Abeam, Cambridge, Mass.

In embodiments, SIRT1, SIRT3 and SIRT6 expression (e.g., mRNA orprotein) in a sample (e.g., cells or tissues in vivo or in vitro)treated using an antisense oligonucleotide of the invention is evaluatedby comparison with Sirtuin (SIRT) expression in a control sample. Forexample, expression of the protein or nucleic acid can be compared usingmethods known to those of skill in the art with that in a mock-treatedor untreated sample. Alternatively, comparison with a sample treatedwith a control antisense oligonucleotide (e.g., one having an altered ordifferent sequence) can be made depending on the information desired. Inanother embodiment, a difference in the expression of the Sirtuin (SIRT)protein or nucleic acid in a treated vs. an untreated sample can becompared with the difference in expression of a different nucleic acid(including any standard deemed appropriate by the researcher, e.g., ahousekeeping gene) in a treated sample vs. an untreated sample.

Observed differences can be expressed as desired, e.g., in the form of aratio or fraction, for use in a comparison with control. In embodiments,the level of a Sirtuin (SIRT) mRNA or protein, in a sample treated withan antisense oligonucleotide of the present invention, is increased ordecreased by about 1.25-fold to about 10-fold or more relative to anuntreated sample or a sample treated with a control nucleic acid. Inembodiments, the level of a Sirtuin (SIRT) mRNA or protein is increasedor decreased by at least about 1.25-fold, at least about 1.3-fold, atleast about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold,at least about 1.7-fold, at least about 1.8-fold, at least about 2-fold,at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold,at least about 4-fold, at least about 4.5-fold, at least about 5-fold,at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold,at least about 7-fold, at least about 7.5-fold, at least about 8-fold,at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold,or at least about 10-fold or more.

Kits, Research Reagents, Diagnostics, and Therapeutics

The compounds of the present invention can be utilized for diagnostics,therapeutics, and prophylaxis, and as research reagents and componentsof kits. Furthermore, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the fimetion of particular genes orto distinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics and in various biological systems, thecompounds of the present invention, either alone or in combination withother compounds or therapeutics, are useful as tools in differentialand/or combinatorial analyses to elucidate expression patterns of aportion or the entire complement of genes expressed within cells andtissues.

As used herein the term “biological system” or “system” is defined asany organism, cell, cell culture or tissue that expresses, or is madecompetent to express products of the Sirtuin (SIRT). These include, butare not limited to, humans, transgenic animals, cells, cell cultures,tissues, xenografts, transplants and combinations thereof.

As one non limiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundsthat affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, (2000) FEBS Lett., 480,17-24; Celis, et al., (2000) FEBS Lett., 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., (2000) Drug Discov. Today,5, 415-425), READS (restriction enzyme amplification of digested eDNAs)(Prashar and Weissman, (1999) Methods Enzymol, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., (2000) Proc. Natl. Acad.Set. USA., 97, 1976-81), protein arrays and proteomics (Cells, et al.,(2000) FEBS Lett., 480, 2-16; Jungblut, et at, Electrophoresis, 1999,20, 2100-10), expressed sequence tag (PST) sequencing (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80,143-57), subtractive RNA fingerprinting (SURF) (Fuchs, et at, (2000)Anal. Biochem. 286, 91-98; Larson, et al., (2000) Cytomeiry 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, (2000) Curr. Opin. Microbial. 3, 316-21), comparative genomichybridization (Carulli, et al., (1998) J. Cell Biochem. Suppl., 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, (1999) Eur. J. Cancer, 35, 1895-904) and mass spectrometrymethods (To, Comb. (2000) Chem, High Throughput Screen, 3, 235-41).

The compounds of the invention are useful for research and diagnostics,because these compounds hybridize to nucleic acids encoding a Sirtuin(SIRT). For example, oligonucleotides that hybridize with suchefficiency and under such conditions as disclosed herein as to beeffective Sirtuin (SIRT) modulators are effective primers or probesunder conditions favoring gene amplification or detection, respectively.These primers and probes are useful in methods requiring the specificdetection of nucleic acid molecules encoding a Sirtuin (SIM) and in theamplification of said nucleic acid molecules for detection or for use infurther studies of a Sirtuin (SIRT). Hybridization of the antisenseoligonucleotides, particularly the primers and probes, of the inventionwith a nucleic acid encoding a Sirtuin (SIRT) can be detected by meansknown in the art. Such means may include conjugation of an enzyme to theoligonucleotide, radiolabeling of the oligonucleotide, or any othersuitable detection means. Kits using such detection means for detectingthe level of a Sirtuin (SIRT) in a sample may also be prepared.

The specificity and sensitivity of antisense are also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs have beensafely and effectively administered to humans and numerous clinicaltrials are presently underway. It is thus established that antisensecompounds can be useful therapeutic modalities that can be configured tobe useful in treatment mimes for the treatment of cells, tissues andanimals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder which can be treated by modulating the expression ofa Sirtuin (SIRT) polynucleotide is treated by administering antisensecompounds in accordance with this invention. For example, in onenon-limiting embodiment, the methods comprise the step of administeringto the animal in need of treatment, a therapeutically effective amountof a Sirtuin (SIRT) modulator. The Sirtuin (SIRT) modulators of thepresent invention effectively modulate the activity of a Sirtuin (SIRT)or modulate the expression of a Sirtuin (SIRT) protein. In oneembodiment, the activity or expression of a Sirtuin (SIRT) in an animalis inhibited by about 10% as compared to a control. Preferably, theactivity or expression of a Sirtuin (SIRT) in an animal is inhibited byabout 30%. More preferably, the activity or expression of a Sirtuin(SIRT) in an animal is inhibited by 50% or more. Thus, the oligomericcompounds modulate expression of a Sirtuin (SIRT) mRNA by at least 10%,by at least 50%, by at least 25%, by at least 30%, by at least 40%, byat least 50%, by at least 60%, by at least 70%, by at least 75%, by atleast 80%, by at least 85%, by at least 90%, by at least 95%, by atleast 98%, by at least 99%, or by 100% as compared to a control.

In one embodiment, the activity or expression of a Sirtuin (SIRT) and/orin an animal is increased by about 10% as compared to a control.Preferably, the activity or expression of a Sirtuin (SIRT) in an animalis increased by about 30%. More preferably, the activity or expressionof a Sirtuin (SIRT) in an animal is increased by 50% or more. Thus, theoligomeric compounds modulate expression of a Sirtuin (SIRT) mRNA by atleast 10% by at least 50%, by at least 25%, by at least 30%, by at least40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%,by at least 80%, by at least 85%, by at least 90%, by at least 95%, byat least 98%, by at least 99%, or by 100% as compared to a control.

For example, the reduction of the expression of a Sirtuin (SIRT) may bemeasured in serum, blood, adipose tissue, liver or any other body fluid,tissue or organ of the animal. Preferably, the cells contained withinsaid fluids, tissues or organs being analyzed contain a nucleic acidmolecule encoding Sirtuin (SIRT) peptides and/or the Sirtuin (SIRT)protein itself.

The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the compounds andmethods of the invention may also be useful prophylactically.

Conjugates:

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. These moieties or conjugates can includeconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typicalconjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed inInternational Patent Application No. PCT/US92109196, filed Oct. 23,1992, and U.S. Pat. No. 6,287,860, which am incorporated herein byreference. Conjugate moieties include, but are not limited to, lipidmoieties such as a cholesterol moiety, cholic acid, a thioether, e.g.,hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic.

Representative United States patents that teach the preparation of sucholigonucleotides conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,46,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

Formations:

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,165; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

Although, the antisense oligonucleotides do not need to be administeredin the context of a vector in order to modulate a target expressionand/or function, embodiments of the invention relates to expressionvector constructs for the expression of antisense oligonucleotides,comprising promoters, hybrid promoter gene sequences and possess astrong constitutive promoter activity, or a promoter activity which canbe induced in the desired case.

In an embodiment, invention practice involves administering at least oneof the foregoing antisense oligonucleotides with a suitable nucleic aciddelivery system. In one embodiment, that system includes a non-viralvector operably linked to the polynucleotide. Examples of such nonviralvectors include the oligonucleotide alone (e.g. any one or more of SEQID NOS: 15 to 94) or in combination with a suitable protein,polysaccharide or lipid formulation.

Additionally suitable nucleic acid delivery systems include viralvector, typically sequence from at least one of an adenovirus,adenovirus-associated virus (AAV), helper-dependent adenovirus,retrovirus, or hemagglutinatin virus of Japan-liposome (HVJ) complex.Preferably, the viral vector comprises a strong eukaryotic promoteroperably linked to the polynucleotide e.g., a cytomegalovirus (CMV)promoter.

Additionally vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include Moloney murine leukemia virusesand HIV-based viruses. One HIV based viral vector comprises at least twovectors wherein the gag and poi genes are from an HIV genome and the envgene is from another virus. DNA viral vectors are preferred. Thesevectors include pox vectors such as orthopox or avipox vectors,herpesvirus vectors such as a herpes simplex 1 virus (HSV) vector,Adenovirus Vectors and Adeno-associated Virus Vectors).

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligonucleotides, examples of pharmaceutically acceptable salts andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein by reference.

The present invention also includes pharmaceutical compositions andformulations that include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer, intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration.

For treating tissues in the central nervous system, administration canbe made by, e.g., injection or infusion into the cerebrospinal fluid.Administration of antisense RNA into cerebrospinal fluid is described,e.g., in U.S. Pat. App. Pub. No. 2007/0117772, “Methods for slowingfamilial ALS disease progression,” incorporated herein by reference inits entirety.

When it is intended that the antisense oligonucleotide of the presentinvention be administered to cells in the central nervous system,administration can be with one or more agents capable of promotingpenetration of the subject antisense oligonucleotide across theblood-brain barrier. Injection can be made, e.g., in the entorhinalcortex or hippocampus. Delivery of neurotrophic factors byadministration of an adenovirus vector to motor neurons in muscle tissueis described in, e.g., U.S. Pat. No. 6,632,427,“Adenoviral-vector-mediated gene transfer into medullary motor neurons,”incorporated herein by reference. Delivery of vectors directly to thebrain, e.g., the striatum, the thalamus, the hippocampus, or thesubstantia nigra, is known in the art and described, e.g., in U.S. Pat.No. 6,756,523, “Adenovirus vectors for the transfer of foreign genesinto cells of the central nervous system particularly in brain,”incorporated herein by reference. Administration can be rapid as byinjection or made over a period of time as by slow infusion oradministration of slow release formulations.

The subject antisense oligonucleotides can also be linked or conjugatedwith agents that provide desirable pharmaceutical or pharmacodynamicproperties. For example, the antisense oligonucleotide can be coupled toany substance, known in the art to promote penetration or transportacross the blood-brain bather, such as an antibody to the transferrinreceptor, and administered by intravenous injection. The antisensecompound can be linked with a viral vector, for example, that makes theantisense compound more effective and/or increases the transport of theantisense compound across the blood-brain barrier. Osmotic blood brainbarrier disruption can also be accomplished by, e.g., infusion of sugarsincluding, but not limited to, meso erythritol, xylitol, D(+) galactose,D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(−) fructose, D(−)marmitol, D(+) glucose, D(+) arabinose, D(−) arabinose, cellobiose, D(+)maltose, D(+) raffinose, rhamnose, D(+) melibiose, D(−) ribose,adonitol, D(+) arabitol, L(−) arabitol, D(+) fucose, L(−) fucose, D(−)lyxose, I(+) lyxose, and L(−) lyxose, or amino acids including, but notlimited to, glutamine, lysine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glycine, histidine, leucine, methionine,phenylalanine, proline, serine, threonine, tyrosine, valine, andtaurine. Methods and materials for enhancing blood brain barrierpenetration are described, e.g., in U.S. Pat. No. 4,866,042, “Method forthe delivery of genetic material across the blood brain barrier,” U.S.Pat. No. 6,294,520, “Material for passage through the blood-brainbarrier,” and U.S. Pat. No. 6,936,589, “Parenteral delivery systems,”all incorporated herein by reference in their entirety.

The subject antisense compounds may be admixed, encapsulated, conjugatedor otherwise associated with other molecules, molecule structures ormixtures of compounds, for example, liposomes, receptor-targetedmolecules, oral, rectal, topical or other formulations, for assisting inuptake, distribution and/or absorption. For example, cationic lipids maybe included in the formulation to facilitate oligonucleotide uptake. Onesuch composition shown to facilitate uptake is LIPOFECTIN (availablefrom GIBCO-BRL, Bethesda, Md.).

Oligonucleotides with at least one 2′-O-methoxyethyl modification arebelieved to be particularly useful for oral administration.Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carriers) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into anymany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances that increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogeneous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug that may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes that are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids. When incorporated into liposomes, these specialized lipidsresult in liposomes with enhanced circulation lifetimes relative toliposomeslacking such specialized lipids. Examples of stericallystabilized liposomes are those in which part of the vesicle-forminglipid portion of the liposome comprises one or more glycolipids or isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. Liposomes and their uses are furtherdescribed in U.S. Pat. No. 6,287,860.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein by reference.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating nonsurfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein by reference.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

formulations for topical administration include those in which theoligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants lipids and liposomesinclude neutral (e.g. dioleoyl-phosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoyl-phosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids bay acids andesters, pharmaceutically acceptable salts thereof, and their uses arefurther described in U.S. Pat. No. 6,287,860.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable, oral formulations are thosein which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants anddictators surfactants include fatty acids and/or esters or saltsthereof, bile acids and/or salts thereof bile acids/salts and fattyacids and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein by reference. Also are combinations ofpenetration enhances, for example, fatty acids/salts in combination withbile acids/salts. A particularly combination is the sodium salt oflaurie acid, cupric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally, in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein by reference.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherchemotherapeutic agents that function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited tocancer chemotherapeutic drugs such as daunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bischloroethyl-nitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurca,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclo-phosphoramide, 5-fluorouracil (5-FU),5-fluotndeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, enrol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andOligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. For example, the first targetmay be a particular antisense sequence of a Sirtuin (STRT), and thesecond target may be a region from another nucleotide sequence.Alternatively, compositions of the invention may contain two or moreantisense compounds targeted to different regions of the same Sirtuin(SIRT) nucleic acid target. Ntunerous examples of antisense compoundsare illustrated herein and others may be selected from among suitablecompounds known in the at Two or more combined compounds may be usedtogether or sequentially.

Dosing:

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC50s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 μgto 100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily; to once every 20 years.

In embodiments, a patient is treated with a dosage of drug that is atleast about 1, at least about 2, at least about 3, at least about 4, atleast about 5, at least about 6, at least about 7, at least about 8, atleast about 9, at least about 10, at least about 15, at least about 20,at least about 25, at least about 30, at least about 35, at least about40, at least about 45, at least about 50, at least about 60, at leastabout 70, at least about 80, at least about 90, or at least about 100mg/kg body weight. Certain injected dosages of antisenseoligonucleotides are described, e.g., in U.S. Pat. No. 7,563,884,“Antisense modulation of PTPIB expression,” incorporated herein byreference in its entirety.

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not limitation. Numerous changes to the disclosedembodiments can be made in accordance with the disclosure herein withoutdeparting from the spirit or scope of the invention. Thus, the breadthand scope of the present invention should not be limited by any of theabove described embodiments.

All documents mentioned herein are incorporated herein by reference. Allpublications and patent documents cited in this application areincorporated by reference for all purposes to the same extent as if eachindividual publication or patent document were so individually denoted.By their citation of various references in this document, Applicants donot admit any particular reference is “prior art” to their invention.Embodiments of inventive compositions and methods are illustrated in thefollowing examples.

EXAMPLES

The following non-limiting Examples serve to illustrate selectedembodiments of the invention. It will be appreciated that variations inproportions and alternatives in elements of the components shown will beapparent to those skilled in the art and are within the scope ofembodiments of the present invention.

Example 1 Design of Antisense Oligonucleotides Specific for a NucleicAcid Molecule Antisense to a Sirtuin (SIRT) and/or a Senses Strand of aSirtuin (SIRT) Polynucleotide

As indicated above the term “oligonucleotide specific for” or“oligonucleotide targets” refers to an oligonucleotide having a sequence(i) capable of forming a stable complex with a portion of the targetedgene, or (ii) capable of forming a stable duplex with a portion of anmRNA transcript of the targeted gene.

Selection of appropriate oligonucleotides is facilitated by usingcomputer programs that automatically align nucleic acid sequences andindicate regions of identity or homology. Such programs are used tocompare nucleic acid sequences obtained, for example, by searchingdatabases such as GenBank or by sequencing PCR products. Comparison ofnucleic acid sequences from a range of species allows the selection ofnucleic acid sequences that display an appropriate degree of identitybetween species. In the case of genes that have not been sequenced,Southern blots are performed to allow a determination of the degree ofidentity between genes in target species and other species. Byperforming Southern blots at varying degrees of stringency, as is wellknown in the art, it is possible to obtain an approximate measure ofidentity. These procedures allow the selection of oligonucleotides thatexhibit a high degree of complementarity to target nucleic acidsequences in a subject to be controlled and a lower degree ofcomplementarity to corresponding nucleic acid sequences in otherspecies. One skilled in the art will realize that there is considerablelatitude in selecting appropriate regions of genes for use in thepresent invention.

An antisense compound is “specifically hybridizable” when binding of thecompound to the and nucleic acid interferes with the normal function ofthe target nucleic acid to cause a modulation of function and/oractivity, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target nucleicacid sequences under conditions in which specific binding is desired,i.e., under physiological conditions in the case of in vivo assays ortherapeutic treatment, and under conditions in which assays areperformed in the case of in vitro assays

The hybridization properties of the oligonucleotides described hereincan be determined by one or more in vitro assays as known in the art.For example, the properties of the oligonucleotides described herein canbe obtained by determination of binding strength between the targetnatural antisense and a potential drug molecules using melting curveassay.

The binding strength between the target natural antisense and apotential drug molecule (Molecule) can be estimated using any of theestablished methods of measuring the strength of intermolecularinteractions, for example, a melting curve assay.

Melting curve assay determines the temperature at which a rapidtransition from double-stranded to single-stranded conformation occursfor the natural antisense/Molecule complex. This temperature is widelyaccepted as a reliable measure of the interaction strength between thetwo molecules.

A melting curve assay can be performed using a cDNA copy of the actualnatural antisense RNA molecule or a synthetic DNA or RNA nucleotidecorresponding to the binding site of the Molecule. Multiple kitscontaining all necessary reagents to perform this assay are available(e.g. Applied Biosystems Inc. MeltDoctor kit). These kits include asuitable buffer solution containing one of the double strand. DNA(dsDNA) binding dyes (such as ABI HRM dyes, SYBR Green, SYTO, etc). Theproperties of the dsDNA dyes are such that they emit almost nofluorescence in free form, but are highly fluorescent when hound todsDNA.

To perform the assay the cDNA or a corresponding oligonucleotide aremixed with Molecule in concentrations defined by the particularmanufacturer's protocols. The mixture is heated to 95° C. to dissociateall pre-formed dsDNA complexes, then slowly cooled to room temperatureor other lower temperature defined by the kit manufacturer to allow theDNA molecules to anneal. The newly formed complexes are then slowlyheated to 95° C. with simultaneous continuous collection of data on theamount of fluorescence that is produced by the reaction. Thefluorescence intensity is inversely proportional to the amounts of dsDNApresent in the reaction. The data can be collected using a real time PCRinstrument compatible with the kit (e.g. ABI's StepOne Plus Real TimePCR System or LightTyper instrument, Roche Diagnostics, Lewes, UK).

Melting peaks are constructed by plotting the negative derivative offluorescence with respect to temperature (-d(Fluorescence)/dT) on they-axis) against temperature (x-axis) using appropriate software (forexample LightTyper (Roche) or SDS Dissociation Curve, ABU The data isanalyzed to identify the temperature of the rapid transition from dsDNAcomplex to single strand molecules. This temperature is called Tm and isdirectly proportional to the strength of interaction between the twomolecules. Typically, Tm will exceed 40° C.

Example 2 Modulation of SIRT Polynticleotides

Treatment of HepG2 Cells with Antisense Oligonucleotides

HepG2 cells from ATCC (cat# HB-8065) were grown in growth media(MEM/EBSS (Hyclone cat # SH30024, or Mediatech cat # MT-10-010-CV)+10%FBS (Mediatech cat# MT35-011-CV)+penicillin/streptomycin (Mediatech cat#MT30-002-CI)) at 37° C. and 5% CO₂. One day before the experiment thecells were replated at the density of 1.5×10⁵/ml into 6 well plates andincubated at 37° C. and 5% CO₂. On the day of the experiment the mediain the 6 well plates was changed to fresh growth media. All antisenseoligonucleotides were diluted to the concentration of 20 μM. Two μl ofthis solution was incubated with 400 μl of Opti-MEM media (Gihcocat#31985-070) and 4 μl of Lipofectamine 2000 (Invitrogen cat#11668019)at room temperature for 20 min and applied to each well of the 6 wellplates with HepG2 cells. A Similar mixture including 2 μl of waterinstead of the oligonucleotide solution was used for themock-transfected controls. After 3-18 h of incubation at 37° C. and 5%CO₂ the media was changed to fresh growth media. 48 h after addition ofantisense oligonucleotides the media was removed and RNA was extractedfrom the cells using SV Total RNA Isolation System from Promega(cat#Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat#74181)following the manufacturers' instructions. 600 ng of RNA was added tothe reverse transcription reaction performed using Verso cDNA kit fromThermo Scientific (cat#AB1453B) or High Capacity cDNA ReverseTranscription Kit (cat#4368813) as described in the manufacturer'sprotocol. The cDNA from this reverse transcription reaction was used tomonitor gene expression by real time PCR using ABI Taqman GeneExpression Mix (cat#4369510) and primers/probes designed by ABI(Applied. Biosystems ragman Gene Expression Assay: Hs00202021_ml,Hs00202030_ml and Hs00213036_ml by Applied Biosystems Inc., Foster CityCalif.), The following PCR cycle was used: 50° C. for 2 min, 95° C. for10 min, 40 cycles of 95° C. for 0.15 seconds, 60° C. for 1 min) usingStepOne Plus Real Time PCR Machine (Applied Biosystems).

Fold change in acne expression after treatment with antisenseoligonucleotides was calculated based on the difference in18S-normalized dCt values between treated and mock-transfected samples.

Results:

Real time PCR results show that the levels of the SIRT1 mRNA in HepG2cells significantly increased 48 h after treatment with some antisenseoligonucleotides to SIRT1 antisense CV396200 (FIG. 3, 4).

Real Time PCR results show that levels of SIRT1 mRNA in HepG2 cells aresignificantly increased in one of the oligonucleotides designed to SIRT1antisense CV396200 (FIG. 8).

Real Time PCR results show that levels of SIRT1 mRNA in HepG2 cells aresignificantly increased in two of the oligonucleotides designed to SIRT1antisense CV428275 (FIG. 9).

The results show that a significant increase in SIRT1 mRNA levels inHepG2 cells 48 hours after treatment with one of the oligonucleotidesdesigned to SIRT antisense BE717453. (FIG. 10).

The results show that show that the levels of the SIRT1 mRNA in HepG2cells are significantly increased 48 h after treatment with three of theoligonucleotides designed to SIRT1 antisense AV718812 respectively (FIG.11).

Real time PCR results show that the levels of SIRT1 mRNA in HepG2 cellsare significantly increased 48 h after treatment with two of the oligosdesigned to SIRT1 antisense AW169958 (FIG. 12).

RT PCR results show that sirt3 levels in HepG2 cells are increased 48hours after treatment with phosphorothioate antisense oligonucleotidesdesigned to sirt3 antisense Hs.683117 (CUR-1545-1550) (FIG. 17).

Real time PCR results show that the levels of SIRT6 mRNA in HepG2 cellsare significantly increased 48 h after treatment with one of the oliogsdesigned to SIRT6 antisense NM_(—)133475 (FIG. 18).

Real time PCR results show that the levels of SIRT6 mRNA in HepG2 cellsare significantly increased 48 h after treatment with one of the oliogsdesigned to SIRT6 antisense bf772662 (FIG. 19)

Treatment of 373 Cells with Antisense Oligonucleotides

3T3 cells from ATCC (cat# CRL-1658) were grown in growth media (MEM/EBSS(Hyclone cat #SH30024, or Mediatech cat #MT-10-010-CV) +10% FBS(Mediatech cat#MT35-011-CV)+penicillin/streptomycin (Mediatechcat#MT30-002-CI)) at 37° C. and 5% CO₂. One day before the experimentthe cells were replated at the density of 1.5×10⁵/ml into 6 well platesand incubated at 37° C. and 5% CO₂. On the day of the experiment themedia in the 6 well plates was changed to flesh growth media. Allantisense oligonucleotides were diluted to the concentration of 20 μM.Two μl of this solution was incubated with 400 μl of Opti-MEM mediaGibco cat#31985-070) and 4 μl of Lipofectamine 2000 (Invitrogencat#11668019) at room temperature for 20 min and applied to each well ofthe 6 well plates with 3T3 cells. A Similar mixture including 2 μl ofwater instead of the oligonucleotide solution was used for themock-transfected controls. After 3-18 h of incubation at 37° C. and 5%CO₂ the media was changed to fresh growth media. 48 h after addition ofantisense oligonucleotides the media was removed and RNA was extractedfrom the cells using SV Total RNA Isolation System from Promega(cat#Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat#74181)following the manufacturers' instructions. 600 ng of RNA was added tothe reverse transcription reaction performed using Verso cDNA kit fromThermo Scientific (cat#AB1453B) or High Capacity cDNA ReverseTranscription Kit (cat#4368813) as described in the manufacturer'sprotocol. The cDNA from this reverse transcription reaction was used tomonitor gene expression by real time PCR using ABI Taqman GeneExpression Mix (cat#4369510) and primers/probes designed by ABI (AppliedBiosystems Taqman Gene Expression Assay: Hs00202021_ml by AppliedBiosystems Inc., Foster City Calif.). The following PCR cycle was used:50° C. for 2 min, 95° C. for 10 min, 40 cycles of (95° C. for 0.15seconds, 60° C. for 1 min) using StepOne Plus Real Time PCR Machine(Applied Biosystems).

Fold change in acne expression after treatment with antisenseoligonucleotides was calculated based on the difference in18S-normalized dCt values between treated and mock-transfected samples.

Results:

Real time PCR results show that the levels of SIRT1 mRNA aresignificantly increased in 313 cells 48 h after treatment with three ofthe oligonucleotides designed to SIRT1 mouse antisense AK044604 (FIG.13).

Real time PCR results show that the levels of SIRT1 mRNA aresignificantly increased in 3T3 cells 48 h after treatment with five ofthe oligonucleotides designed to SIRT1 mouse antisense AK044604 (FIG.14).

Real time PCR results show that the levels of SIRT1 mRNA aresignificantly increased in 3T3 cells 48 h after treatment with two ofthe oligonucleotides designed to SIRT1 mouse antisense AK044604 (FIG.15).

Real time PCR results show that the levels of SIRT1 mRNA aresignificantly increased in 3T3 cells 48 h after treatment with two ofthe oligonucleotides designed to SIRT1 mouse antisense AK044604 (FIG.16).

Treatment of Vero76 Cells with Antisense Oligonucleotides:

Vero76 cells from ATCC (cat# CRL-1587) were grown in growth media(MEM/EBSS (Hyclone cat #SH30024, or Mediatech cat #MT-10-010-CV) +10%FBS (Mediatech cat# MT35-011-CV)+penicillin/streptomycin (Mediatech cat#MT30-002-CI)) at 37° C. and 5% CO2. One day before the experiment thecells were replated at the density of 1.5×10⁵/ml into 6 well plates andincubated at 37° C. and 5% CO2. On the day of the experiment the mediain the 6 well plates was changed to fresh growth media. All antisenseoligonucleotides were diluted in water to the concentration of 20 μM. 2μl of this solution was incubated with 400 μl of Opti-MEM media (Cawcat#31985-070) and 4 μl of Lipofectamine 2000 (Invitrogen cat#11668019)at room temperature for 20 min and applied to each well of the 6 wellplates with Vero76 cells. Similar mixture including 2 μl of waterinstead of the oligonucleotide solution was used for themock-transfected controls. After 3-18 h of incubation at 37° C. and 5%CO2 the media was changed to fresh growth media. 48 h after addition ofantisense oligonucleotides the media was removed and RNA was extractedfrom the cells using SV Total RNA Isolation System from Promega (cat#Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat#74181),following the manufacturers' instructions. 600 ng of RNA was added tothe reverse transcription reaction performed using Verso cDNA kit fromThermo Scientific (cateAB1453B) as described in the manufacturer'sprotocol. The cDNA from this reverse transcription reaction was used tomonitor gene expression by real time PCR using ABI Taqman GeneExpression Mix (cat#4369510) and primers/probes designed by ABI (AppliedBiosystems Taqman Gene Expression Assay: Hs 00202021_ml by AppliedBiosystems Inc., Foster City Calif.). The following PCR cycle was used:50° C. for 2 min, 95° C. for 10 min, 40 cycles of (95° C. for 15seconds, 60° C. for 1 min) using StepOne Plus Real Time PCR Machine(Applied Biosystems). Fold change in gene expression after treatmentwith antisense oligonucleotides was calculated based on the differencein 18S-normalized dCt values between treated and mock-transfectedsamples.

Results:

Real time PCR results show that the levels of the SIRT1 mRNA in Verocells significantly increased 48 h after treatment with antisenseoligonucleotides to SIRT1 antisense CV396200 (FIG. 5).

Treatment of DBS Cells with Antisense Oligonucleotides

DBS cells from ATCC (cat#CCL-161) were grown in growth media (MEM/EBSS(Hyclone cat #SH30024, or Mediatech cat #MT-10-010-CV) +10% FBS(Mediatech cat MT35-011-CV)+penicillin/streptomycin (Mediatechcat#MT30-002-CI)) at 37° C. and 5% CO₂. One day before the experimentthe cells were replated at the density of 15×10⁵/ml into 6 well platesand incubated at 37° C. and 5% CO₂. On the day of the experiment themedia in the 6 well plates was changed to fresh growth media. Allantisense oligonucleotides were diluted to the concentration of 20 μM.Two μl of this solution was incubated with 400 μl of Opti-MEM media(Gibco cat#31985-070) and 4 μl of Lipofecutmine 2000 (Invitrogencat#11668019) at mom temperature for 20 min and applied to each well ofthe 6 well plates with 3T3 cells. A. Similar mixture including 2 μl ofwater instead of the oligonucleotide solution was used for themock-transfected controls. After 3-18 h of incubation at 37° C. and 5%CO₂ the media was changed to fresh growth media. 48 h after addition ofantisense oligonucleotides the media was removed and RNA was extractedfrom the cells using SV Total RNA Isolation System from Promega(cat#Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat#74181)following the manufacturers' instructions. 600 ng of RNA was added tothe reverse transcription reaction performed using Verso cDNA kit fromThermo Scientific (cat#AB1453B) or High Capacity cDNA ReverseTranscription Kit (cat#4368813) as described in the manufacturer'sprotocol. The cDNA from this reverse transcription reaction was used tomonitor gene expression by real time PCR using ABI Tacpnan GeneExpression Mix (cat#4369510) and primers/probes designed by ABI (AppliedBiosystems Taqman Gene Expression Assay: Hs00213036_ml by AppliedBiosystems Inc., Foster City Calif.). The following PCR cycle was used:50° C. for 2 min, 95° C. for 10 min, 40 cycles of (95° C. for 15seconds, 60° C. for 1 min) using StepOne Plus Real Time PCR Machine(Applied Biosystems).

Fold change in gene expression after treatment with antisenseoligonucleotides was calculated based on the difference in18S-normalized dCt values between treated and mock-transfected samples.

Results:

Real time PCR results show that the levels of SIRT6 mRNA in DBS cellsare significantly increased 48 h after treatment with two of the oliogsdesigned to SIRT6 antisense bf772662 and one oligo designed toNM_(—)133475 (FIG. 20).

Example 3 Modulation of SIRT Gene Expression Materials and Methods

Treatment of HepG2 Cells with Naked Antisense Oligonucleotides

HepG2 cells from ATCC (cat#HB-8065) were grown in growth media (MEM/EBSS(Hyclone cat#SH30024, or Mediatech cat #MT-10-010-CV) +10% FBS(Mediatech cat# MT35-011-CV)+penicillin/streptomycin (Mediatech cat#MT30-002-CI)) at 37° C. and 5% CO2. One day before the experiment thecells were replaced at the density of 0.5×10⁵/ml into 6 well plates andincubated at 37° C. and 5% CO2. On the day of the experiment the mediain the 6 well plates was replaced with 1.5 ml/well of fresh growthmedia. All antisense oligonucleotides were diluted in water to theconcentration of 20 μM. 2 μl of this solution was incubated with 400 μlof Opti-MEM media (Gibco cat#31985-070) and 4 ul of Lipofectamine 2000(Invitrogen cat#11668019) at room temperature for 20 min and applied toeach well of the 6 well plates with HepG2 cells. Similar mixtureincluding 2 μl of water instead of the oligonucleotide solution was usedfor the mock-transfected controls. After 3-18 h of incubation at 37° C.and 5% CO2 the media was changed to fresh growth media. 72 h afteraddition of antisense oligonucleotides the cells were redosed asdescribed in above. 48 h after the second dosing of antisenseoligonucleotides the media was removed and RNA was extracted from thecells using SV Total RNA Isolation System from Promega (cat #Z3105) orRNeasy Total RNA isolation kit from Qiagen (cat#74181) following themanufacturers' instructions. 600 ng of RNA was added to the reversetranscription reaction performed using Verso cDNA kit from ThermoScientific (cat#AB145313) as described in the manufacturer's protocol.The cDNA from this reverse transcription reaction was used to monitorgene expression by real time PCR using ABI Taqman Gene Expression Mix(cat#4369510) and primers/probes designed by ABI (Applied BiosystemsTacpuan Gene Expression Assay: Hs00202021_ml, Hs00202030_ml andHs00213036_ml by Applied Biosystems Inc., Foster City Calif.). Thefollowing PCR cycle was used: 50° C. for 2 min, 95° C. for 10 min, 40cycles of (95° C. for 15 seconds, 60° C. for 1 min) using StepOne PlusReal Time PCR. Machine (Applied Biosystems). Fold change in geneexpression after treatment with antisense oligonucleotides wascalculated based on the difference in 18S-normalized dCt values betweentreated and mock-transfected samples.

Primers and probe for the custom designed ragman assay for exon 4:AACTGGAGCTGGGGTGTCTGTTTCA (SEQ ID NO: 95) the SIRT1 natural antisenseCV396200.

Forward Primer Seq. (SEQ ID NO: 96) CCATCAGACGACATCCCTTAACAAAReverse Primer Seq. (SEQ ID NO: 97) ACATTATATCATAGCTCCTAAAGGAGATGCAReporter Seq. (SEQ ID NO: 98) CAGAGTTTCAATTCCC

Results:

The results show that the levels of the SIRT1 mRNA in HepG2 cells aresignificantly increased 48 h after treatment with one of the siRNAsdesigned to sirtas (sirtas_(—)5, P=0.01). In the same samples the levelsof sirtas RNA were significantly decreased after treatment withsirtas_(—)5, but unchanged after treatment with sirtas_(—)6 andsirtas_(—)7, which also had no effect on the SIRT1 mRNA levels (FIG. 2).sinas_(—)5, sirtas_(—)6 and sirtas_(—)7 correspond to SEQ ID NO: 38; 39and 40 respectively.

Treatment of Primary Monkey Hepatocytes

Primary monkey hepatocytes were introduced into culture by RxGen Inc.and plated in 6 well plates. They were treated with oligonucleotides asfollows. The media in the 6 well plates was changed to fresh growthmedia consisting of William's Medium E (Siuma cat#W4128) supplementedwith 5% FBS, 50 U/ml penicillin and 50 ug/ml streptomycin, 4 ug/mlinsulin, 1 uM dexamethasone, 10 ug/ml Fungin (InVivogen, San DiegoCalif.). All antisense oligonucleotides were diluted to theconcentration of 20 μM. 2 μl of this solution was incubated with 400 μlof Opti-MEM media (Gibco cat#31985-070) and 4 μl of Lipofectamine 2000(Invitrogen cat#11668019) at room temperature for 20 min and applied toeach well of the 6 well plates with cells. Similar mixture including 2μl of water instead of the oligonucleotide solution was used for themock-transfected controls. After 3-18 h of incubation at 37° C. and 5%CO2 the media was changed to fresh growth media. 48 h after addition ofantisense oligonucleotides the media was removed and RNA was extractedfrom the cells using SV Total RNA Isolation System from Promega(cat#Z3105) or RNeasy Total RNA isolation kit from Qiagen (cat#74181)following the manufacturers' instructions. 600 ng of RNA was added tothe reverse transcription reaction performed using Verso cDNA kit fromThermo Scientific (cat#AB1453B) as described in the manufacturer'sprotocol. The cDNA from this reverse transcription reaction was used tomonitor gene expression by real time PCR using ABI Taqman GeneExpression Mix (cat#4369510) and primers/probes designed by ABI (AppliedBiosystems Taqman Gene Expression Assay: Hs00202021_ml, Hs00202030_mland Hs00213036_ml by Applied Biosystems Inc., Foster City Calif.). Thefollowing PCR cycle was used: 50° C. for 2 min, 95° C. for 10 min, 40cycles of (95° C. for 15 seconds, 60° C. for 1 min) using Mx4000 thermalcycler (Stratagene). Fold change in gene expression after treatment withantisense oligonucleotides was calculated based on the difference in18S-normalized dCt values between treated and mock-transfected samples.

Results:

The results are shown in FIG. 7. Real time PCR results show an increasein SIRT1 mRNA levels after treatment with an oligonucleotide againstSIRT1 antisense.

Example 4 Efficacy and Attrition of Action Study of CUR 963 in theAfrican Green Monkey

The objective of this study was to assess and compare the effect ofantisense knockdown of the discordant noncoding antisense sequences thatregulate the SERT1 genes following intravenous administration in anonhuman primate model. The antisense oligonucleotide test articlesdesigned to inhibit the SIRT1 regulatory sequences were designated asCUR 963.

CUR 963: (SEQ II) NO: 34) +G*+T*C*T*G*A*T*G*G*+A*+G*+A.CUR 962 (control): (SEQ ID NO: 99) +G*+C*T*A*G*T*C*T*G*+T*+T*+G.

Regulatory Test Guidelines

This study was designed in accordance with accepted toxicologicalprinciples and to comply with International Conference of Harmonization(ICH) Harmonized Tripartite Guidelines (Non-Clinical Safety Studies forthe Conduct of Human Clinical Trials for Pharmaceuticals ICH M3(m), 2000Nov. 9), and generally accepted procedures for the testing oftherapeutic agents.

Test and Control Articles Test Article Identity and Preparation

The test article, CUR-963, is a chemically stabilized antisenseoligonucleotide. The vehicle for intravenous delivery isphosphate-buffered saline (PBS).

Vehicle Characterization

For the PBS vehicle, the composition, batch number, expiry date andstorage conditions (temperature and light/dark) was obtained from thesupplier.

Test Article Storage and Handling

The test substance and vehicle were stored according to the receivedstorage conditions supplied by the Sponsor and manufacturer,accordingly.

Analysis of the Test Article Formulations

Samples of the test article formulation will be cryopreserved foranalysis of the concentration, stability and homogeneity of the testsubstance formulations.

Test System Rationale

The primate is a suitable non rodent species, acceptable to regulatoryauthorities as an indicator of potential hazards, and for whichextensive background data are available. The African green monkeyspecifically is a highly clinically relevant model of multiple humanphysiologic and disease states.

The intravenous route of administration corresponds to a possible humantherapeutic route. The dose of the test articles was based on theresults of the dose finding studies of analogous compounds previouslyperformed in the African green monkey.

African green monkeys were chosen as the primate of choice as the testsubstances' target sequences are conserved across species with 100%homology in primates. Additionally, the test substance is a syntheticoligonucleotide. Consequently, dosing in primates allows for a superiorassessment of the efficacy of these compounds that would be morereflective of the uptake likely to be seen in humans than in any otherspecies.

Animals

Species:

Chlorocebus scthaeus, non-human primate

Breed:

African green monkey indigenous to St. Kitts.

Source:

RxGcn, Lower Bourryeau, St. Kitts, West Indies.

Expected Age:

The test animals were adults.

Expected Weight.

The monkeys weigh approximately 3-4 kg. The actual range may vary butwill be documented in the data.

Sex:

The test animals were adult females.

Number of Animals:

Ten animals were screened to ensure identification of 8 animalsappropriate for enrollment in the study.

Number on Study:

Females: 8

Justification for Number on Study:

This study was designed to use the fewest number of animals possible,consistent with the primary objective of evaluating the therapeuticefficacy of the test article in the African green monkey and priorstudies of the systemic administration of this type of oligonucleotidein this species.

Animal Specification:

Ten adult African Green monkeys in the weight range of 3 to 4 kg, wereemployed in the study. The monkeys were drug-naïve adult animalshumanely trapped from the feral population that inhabits the island.Trapped monkeys were treated with antihelminthics to eliminate anypossible intestinal parasite burden and were observed in quarantine fora minimum of 4 weeks prior to screening for study enrollment. The age oftrapped monkeys were estimated by size and demotion, with the exclusionof older animals from the study. Prior to study enrollment, a clinicalexam was performed on each monkey, including evaluation of locomotionand dexterity. Blood samples were taken and sent to An tech Diagnostics(Memphis, Tenn.) for comprehensive clinical chemistries and a completeblood count and lipid profiles (see sections 9.2 and 319567928 forspecifications). Monkeys with abnormal lab values, as determined bycomparison to the established normal range for monkeys in the St. Kittscolony, were excluded from the study. In order to identify 8 monkeysthat satisfy this criterion, 10 monkeys were screened, with thescreening of additional animals as needed. Before study initiation, theselected monkeys will be transferred to individual cages to acclimate toindividual housing for a one-week period. Only animals deemed suitablefor experimentation will be enrolled in the study. The actual (orestimated) age and weight ranges at the start of the study will bedetailed in the raw data and final report.

Animal Health and Welfare:

The highest standards of animal welfare were followed and adhered toguidelines stipulated by the St. Kitts Department of Agriculture and theU.S. Department of Health and Human Services. All studies will beconducted in accordance with these requirements and all applicable codesof practice for the care and housing of laboratory animals. Allapplicable standards for veterinary care, operation, and review ascontained in the NIH Guide for the Care and Use of Animals. The St Kittsfacility maintains an animal research committee that reviews theprotocols and inspects the facilities as required by the Guide. TheFoundation has an approved assurance filed with the Office of LaboratoryAnimal Welfare, as required by the Guide, #A4384-01 (Axion ResearchFoundation/St. Kitts Biomedical Foundation). There are no specialnonhuman primate veterinary care issues and biohazard issues raised bythe research specified in this study.

Housing and Environment:

To allow detection of any treatment-related clinical signs, the animalswere housed individually prior to surgery and postoperatively untilsacrifice. The primate building in which the individual cages weresituated were illuminated entirely by ambient light, which at 17 degreesnorth latitude approximates a 12 hr:12 hr light-dark cycle asrecommended in the U.S. D.H.H.S guidelines. The RxGen primate buildingwas completely ventilated to the outside. Additional air movement wasassured by ceiling fans to maintain a constant target temperature of23-35° C., as is typical of St. Kitts throughout the year. Twenty-fourhour extremes of temperature and relative humidity (which also will notbe controlled) were measured daily. During the study, the cages werecleaned at regular intervals.

Diet and Water:

Each animal was offered approximately 90 grams per day of a standardmonkey chow diet (TekLad, Madison, Wis.). The specific nutritionalcomposition of the diet was recorded. The water was periodicallyanalyzed for microbiological purity. The criteria for acceptable levelsof contaminants in stock diet and water supply were within theanalytical specifications established by the diet manufacturer and theperiodic facility water evaluations, respectively. The water met allcriteria necessary for certification as acceptable for humanconsumption.

Experimental Design

Animal Identification and Randomization:

Allocation was done by means of a stratified randomization procedurebased an body-weight and plasma cholesterol profiles. Prior to and afterallocation to a group, each animal was identified by a tattoo on theabdomen. Tattoos are placed on all colony animals as a means ofidentification in the course of routine health inspections. A cage planwas drawn up to identify the individuals housed within, and individualmonkeys were further identified by a labeled tag attached to theirrespective cage.

Group Sizes, Doses and Identification Numbers:

The animals were assigned to 2 treatment groups, comprised of 4 monkeysin each group. Specific animal identification numbers were provided toeach monkey according to the facility numbering system. This systemuniquely identifies each monkey by a letter followed by a three digitnumber, e.g. Y032.

Route and Frequency of Administration:

Animals were dosed once daily on Days 1, 3, and 5 deliveredintravenously by manual infusion over ˜10 min. The infusion rate will be24 mL/kg/h. The animals were sedated with ketamine and xylazine prior toand during the dosing procedure. A venous catheter (Terumo mini veininfusion set, 20 gauge needle, or similar appropriate infusion set) wasinserted into the saphenous vein. Dosing took place in each monkeybetween 8:00 and 10:00 a.m. shortly after the animals wake and prior tofeeding. A blood sample to assess plasma cholesterol and other lipidlevels as described in Blood Chemistry section below, was collected justprior to each infusion. Blood collection preceded feeding at bothsampling intervals to minimize dietary effects on cholesterolmeasurements.

Clinical Observations:

All visible signs of reaction to treatment were recorded on each day ofdosing. In addition, the animals were examined at least once each weekfor physical attributes such as appearance and general condition.

Body Weights:

Body weights were recorded at weekly intervals during the treatment andpost-treatment periods.

Food Consumption:

Individual food consumption was not quantified. Feeding patterns werehowever monitored and a note made of any major changes.

Mortality and Morbidity:

Mortality and morbidity will be recorded. Any decision regardingpremature sacrifice will be made after consultation with the StudyDirector and with the Sponsor's Monitoring Scientist, if possible.Animals that are found dead or killed prematurely will be subjected tonecropsy with collection of liver, kidney, heart and spleen lung tissuesfed histopathology. In the event of premature sacrifice a blood samplewill also be taken (if possible) and the parameters determined. Animalsthat are found dead after regular working hours will be refrigeratedovernight and necropsies performed at the start of the next working day.If the condition of an animal requires premature sacrifice, it will beeuthanized by intravenous overdose of sodium pentobarbital. All researchis governed by the Principles for Use of Animals. RxGen is required bylaw to comply with the U.S. Department of Health and Human Servicesstandards for primate facility, which dictate the levels of severitythat the procedures within this study, specified as mild, must abide.

Clinical Laboratory Studies

Fat Biopsies:

A subcutaneous fat biopsy was performed on all study monkeys except Y775on study days 26 by tissue extraction through a 1 cm midline incisioninferior to the umbilicus. Biopsies were immediately immersed in alabeled cryotube containing 2 mls of RNAlater (Qiagen) and incubated at4° C. overnight, after which the RNAlater was aspirated and the sampletube flash frozen in liquid nitrogen. Following transportation in liquidnitrogen total RNA was isolated for real-time qPCR of target genes.

Results:

Real time PCR results show an increase in SIRT1 mRNA levels in fatbiopsies from monkeys dosed with CUR-963, an oligonucleotide designed toSIRT1 antisense CV396200.1, compared to monkeys dosed with CUR-962 (SEQID NO.: 99), an oligonucleotide which had no effect on SIRT1 expressionin vitro (designed to ApoA1 antisense DA327409, data not shown). mRNAlevels were determined by real time PCR (FIG. 6).

Example 5 In vivo Modulation of Sirtuin (SIRT) by Antisense DNAOligonucleotides

Treatment with Antisense DNA Oligonucleotides (ASO):

Antisense oligonucleotides (ASO) specific for SIRT1. AS are administeredto C57B1/6J mice which are fed a high fat diet for 12 weeks to induceobesity and diabetes. (Purushotham A. et al., (2009) Cell Metabolism 9,p. 327-338,). The treatment of the mice with ASO will start at the timeof the implementation of the high fat diet. Mice are injected IP once aweek with ASO prepared in normal saline, at a concentration of 5 mg/kg.

Measurements of Body Weight and Food Intake:

Body weight and food intake of mice are measured twice per week, priorto IP injection of the ASO.

Blood Glucose Measurements:

Fed and fasted blood glucose concentrations are measured each week bytaking a sample of blood from the tail vein.

Glucose Tolerance Tests (GTT):

The GTT will be done totally twice per mouse, halfway through the diet(at week 4) and near the end (at week 10) of the high fat diet. The GTTwill inform us about the glucose tolerance of the mice that is thecapacity to rapidly clear a glucose bolus from the blood stream. This isa measure for diabetes. Mice are tasted overnight for 16 hours. Mice areinjected IP glucose 2 g/kg. This translates into a final volume of 0.2ml 30% (w/v) glucose solution for a mouse of 30 g weight. Glucosemeasurements are taken prior to glucose injection and at 5, 15, 30, 60,90 and 120 min post-injection. Glucose is measured by cutting the tailtip 1 mm from the end of the tail under isoflurane anesthesia prior toIP glucose injection. The blood droplet is aspirated into a strip andglucose concentration is measured with a glucometer. The GTT will bedone totally twice per mouse, halfway through the diet (at week 4) andnear the end (at week 10) of the high fat diet. The GTT will inform usabout the glucose tolerance of the mice that is the capacity to rapidlyclear a glucose bolus from the blood stream. This is a measure fordiabetes.

Insulin Tolerance Test (ITT):

Mice are tasted for 6 hours from 9 am til 3 pm. Mice are then injectedIP 0.5-1 U Insulin/kg. The insulin concentration will be adjusted suchthat the final injected volume is 0.1-0.15 ml. Blood glucosemeasurements are taken prior to injection and at 5, 15, 30, 45, and 60minutes post-injection. Blood is collected exactly as described underGTT. In addition to monitoring the glucose levels, the behavior of themice is constantly observed during the ITT. Hypoglycemia can manifest asa change in behavior with the animals becoming very quiet and showingdiscomfort. To prevent hypoglycemia, glucose (1 g/kg) is injected IP ina final volume of 0.1-0.15 ml as soon as the blood glucose concentrationfalls below 50 mg/ml or signs of discomfort are observed.

Blood Collection by Facial Vein Puncture:

Mice are restrained by the scruff of the neck and base of the tail,slightly compressing the blood vessels of the neck through the tautnessof the grip on the neck skin. The sampling site is on the jaw slightlyin front of the angle of the mandible. The skin at the sampling site ispunctured with an 18 G needle or a lancet at a 90° angle until the tipof the needle/lancet just passes through the skin. Blood samples arecollected using microhematocrit tubes. After blood has been collected,the grip on the neck is loosened and pressure is applied at theinsertion site with a gauze sponge to ensure hemostasis. 0.05-0.2 ml ofblood will be collected by this method. This procedure will be performedonly once in week 5 of the high fat diet and eventually in week 12 ifthe intracardiac puncture is not working (see below). Blood hormoneswhich regulate the metabolism of glucose and lipids (such as insulin,adiponeetin and leptin) are measured using commercially available ELISAkits. (e.g., R&D Systems, Minneapolis, Minn., Assay. Pro St. Charles,Mo., Mabtech, Mariemont, Ohio)

Intracardiac Puncture:

At the end of the 12 week high fat diet, mice will be anesthetized bycontinuous isoflurane inhalation. Anesthesia is induced by placing themice in an induction box, which is supplied with isoflurane and oxygen.Mice will be restrained on their back. The heart is punctured with a 27G needle. Following exsanguineation, the head is decapitated to ensuredeath. Tissues (liver, pancreas, white and brown adipose tissue, andskeletal muscle) are collected for further investigations (RNA andprotein measurements and histology). Around 0.5-1 ml of blood will beobtained and used to determine several critical parameters of glucoseand lipid metabolism (glucose, insulin, cholesterol, triglycerides, freefatty acids, leptin, adipokines, corticosteroids, thyroid hormones). Ifdifficulties occur in this method, we will collect blood by facial veinpuncture under isoflurane anesthesia instead (see above).

Although the invention has been illustrated and described with respectto one or more implementations, equivalent alterations and modificationswill occur to others skilled in the art upon the reading andunderstanding of this specification and the annexed drawings. Inaddition, while a particular feature of the invention may have beendisclosed with respect to only one of several implementations, suchfeature may be combined with one or more other features of the otherimplementations as may be desired and advantageous for any given orparticular application.

The Abstract of the disclosure will allow the reader to quicklyascertain the nature of the technical disclosure. It is submitted withthe understanding that it will not be used to interpret or limit thescope or meaning of the following claims.

1. A method of a function of and/or the expression of a Sirtuin (SIRT)polynucleotide in patient cells or tissues in vivo or in vitrocomprising: contacting said cells or tissues with at least one antisenseoligonucleotide 5 to 30 nucleotides in length wherein said at least oneoligonucleotide has at least 50% sequence identity to a reversecomplement of a polynucleotide comprising 5 to 30 consecutivenucleotides within nucleotides 1 to 1028 of SEQ ID NO: 5 or nucleotides1 to 429 of SEQ ID NO: 6, or nucleotides 1 to 156 of SEQ ID NO: 7 ornucleotides 1 to 593 of SEQ ID NO:8, 1 to 373 of SEQ ID NO: 9, 1 to 1713of SEQ ID NO: 10, 1 to 660 of SEQ ID NO:11, 1 to 589 of SEQ ID NO: 12, 1to 428 of SEQ ID NO: 13 and 1 to 4041 of SEQ ID NO: 14; therebymodulating a function of and/or the expression of the Sirtuin (SIRT)polynucleotide in patient cells or tissues in vivo or in vitro.
 2. Amethod of modulating a function of and/or the expression of a Sirtuin(SIRT) polynucleotide in patient cells or tissues in vivo or in vitrocomprising: contacting said cells or tissues with at least one antisenseoligonucleotide 5 to 30 nucleotides in length wherein said at least oneoligonucleotide has at least 50% sequence identity to a reversecomplement of a natural antisense of a Sirtuin (SIRT) polynucleotide;thereby modulating a function of and/or the expression of the Sirtuin(SIRT) polynucleotide in patient cells or tissues in vivo or in vitro.3. A method of modulating a function of and/or the expression of aSirtuin (SIRT) polynucleotide in patient cells or tissues in vivo or invitro comprising: contacting said cells or tissues with at least oneantisense oligonucleotide 5 to 30 nucleotides in length wherein saidoligonucleotide has at least 50% sequence identity to an antisenseoligonucleotide to the Sirtuin (SIRT) polynucleotide; thereby modulatinga function of and/or the expression of the Sirtuin (SIRT) polynucleotidein patient cells or tissues in vivo or in vitro.
 4. A method ofmodulating a function of and/or the expression of a Sirtuin (SIRT)polynucleotide in patient cells or tissues in vivo or in vitrocomprising: contacting said cells or tissues with at least one antisenseoligonucleotide that targets a region of a natural antisenseoligonucleotide of the Sirtuin (SIRT) polynucleotide; thereby modulatinga function of and/or the expression of the Sirtuin (SIRT) polynucleotidein patient cells or tissues in vivo or in vitro.
 5. The method of claim4, wherein a function of and/or the expression of the Sirtuin (SIRT) isincreased in vivo or in vitro with respect to a control.
 6. The methodof claim 4, wherein the at least one antisense oligonucleotide targets anatural antisense sequence of a Sirtuin (SIRT) polynucleotide.
 7. Themethod of claim 4, wherein the at least one antisense oligonucleotidetargets a nucleic acid sequence comprising coding and/or non-codingnucleic acid sequences of a Sirtuin (SIRT) polynucleotide.
 8. The methodof claim 4, wherein the at least one antisense oligonucleotide targetsoverlapping and/or non-overlapping sequences of a Sirtuin (SIRT)polynucleotide.
 9. The method of claim 4, wherein the at least oneantisense oligonucleotide comprises one or more modifications selectedfrom: at least one modified sugar moiety, at least one modifiedinternucleoside linkage, at least one modified nucleotide, andcombinations thereof.
 10. The method of claim 9, wherein the one or moremodifications comprise at least one modified sugar moiety selected from:a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modified sugarmoiety, a 2′-O-alkyl modified sugar moiety, a bicyclic sugar moiety, andcombinations thereof.
 11. The method of claim 9, wherein the one or moremodifications comprise at least one modified internucleoside linkageselected from: a phosphorothioate, 2′-Omethoxyethyl (MOE), 2′-fluoro,alkylphosphonate, phosphorodithioate, alkylphosphonothioate,phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate,carboxymethyl ester, and combinations thereof.
 12. The method of claim9, wherein the one or more modifications comprise at least one modifiednucleotide selected from: a peptide nucleic acid (PNA), a locked nucleicacid (LNA), an arabino-nucleic acid (FANA), an analogue, a derivative,and combinations thereof.
 13. The method of claim 1, wherein the atleast one oligonucleotide comprises at least one oligonucleotidesequences set forth as SEQ ID NOS: 15 to
 94. 14. A method of modulatinga function of and/or the expression of a Sirtuin (SIRT) in mammaliancells or tissues in vivo or in vitro comprising: contacting said cellsor tissues with at least one short interfering RNA (siRNA)oligonucleotide 5 to 30 nucleotides in length, said at least one siRNAoligonucleotide being specific for an antisense polynucleotide of aSirtuin (SIRT) polynucleotide, wherein said at least one siRNAoligonucleotide has at least 50% sequence identity to a complementarysequence of at least about five consecutive nucleic acids of theantisense and/or sense nucleic acid molecule of the Sirtuin (SIRT)polynucleotide; and, modulating a function of and/or the expression of aSirtuin (SIRT) in mammalian cells or tissues in vivo or in vitro. 15.The method of claim 14, wherein said oligonucleotide has at least 80%sequence identity to a sequence of at least about five consecutivenucleic acids that is complementary to the antisense and/or sensenucleic acid molecule of the Sirtuin (SIRT) polynucleotide.
 16. A methodof modulating a function of and/or the expression of a Sirtuin (SIRT) inmammalian cells or tissues in vivo or in vitro comprising: contactingsaid cells or tissues with at least one antisense oligonucleotide ofabout 5 to 30 nucleotides in length specific for noncoding and/or codingsequences of a sense and/or natural antisense strand of a Sirtuin (SIRT)polynucleotide wherein said at least one antisense oligonucleotide hasat least 50% sequence identity to at least one nucleic acid sequence setforth as SEQ ID NOS: 1 to 14; and, modulating the function and/orexpression of the Sirtuin (SIRT) in mammalian cells or tissues in vivoor in vitro.
 17. A synthetic, modified oligonucleotide comprising atleast one modification wherein the at least one modification is selectedfrom: at least one modified sugar moiety; at least one modifiedinternucleotide linkage; at least one modified nucleotide, andcombinations thereof; wherein said oligonucleotide is an antisensecompound which hybridizes to and modulates the function and/orexpression of a Sirtuin (SIRT) in vivo or in vitro as compared to anormal control.
 18. The oligonucleotide of claim 17, wherein the atleast one modification comprises an internucleotide linkage selectedfrom the group consisting of phosphorothioate, alkylphosphonate,phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate,carbonate, phosphate triester, acetamidate, carboxymethyl ester, andcombinations thereof.
 19. The oligonucleotide of claim 17, wherein saidoligonucleotide comprises at least one phosphorothioate internucleotidelinkage.
 20. The oligonucleotide of claim 17, wherein saidoligonucleotide comprises a backbone of phosphorothioate internucleotidelinkages.
 21. The oligonucleotide of claim 17, wherein theoligonucleotide comprises at least one modified nucleotide, saidmodified nucleotide selected from: a peptide nucleic acid, a lockednucleic acid (LNA), analogue, derivative, and a combination thereof. 22.The oligonucleotide of claim 17, wherein the oligonucleotide comprises aplurality of modifications, wherein said modifications comprise modifiednucleotides selected from: phosphorothioate, alkylphosphonate,phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate,carbonate, phosphate triester, acetamidate, carboxymethyl ester, and acombination thereof.
 23. The oligonucleotide of claim 17, wherein theoligonucleotide comprises a plurality of modifications, wherein saidmodifications comprise modified nucleotides selected from: peptidenucleic acids, locked nucleic acids (LNA), analogues, derivatives, and acombination thereof.
 24. The oligonucleotide of claim 17, wherein theoligonucleotide comprises at least one modified sugar moiety selectedfrom: a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modifiedsugar moiety, a 2′-O-alkyl modified sugar moiety, a bicyclic sugarmoiety, and a combination thereof.
 25. The oligonucleotide of claim 17,wherein the oligonucleotide comprises a plurality of modifications,wherein said modifications comprise modified sugar moieties selectedfrom: a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modifiedsugar moiety, a 2′-O-alkyl modified sugar moiety, a bicyclic sugarmoiety, and a combination thereof.
 26. The oligonucleotide of claim 17,wherein the oligonucleotide is of at least about 5 to 30 nucleotides inlength and hybridizes to an antisense and/or sense strand of a Sirtuin(SIRT) polynucleotide wherein said oligonucleotide has at least about20% sequence identity to a complementary sequence of at least about fiveconsecutive nucleic acids of the antisense and/or sense coding and/ornoncoding nucleic acid sequences of the Sirtuin (SIRT) polynucleotide.27. The oligonucleotide of claim 17, wherein the oligonucleotide has atleast about 80% sequence identity to a complementary sequence of atleast about five consecutive nucleic acids of the antisense and/or sensecoding and/or noncoding nucleic acid sequence of the Sirtuin (SIRT)polynucleotide.
 28. The oligonucleotide of claim 17, wherein saidoligonucleotide hybridizes to and modulates expression and/or functionof at least one Sirtuin (SIRT) polynucleotide in vivo or in vitro, ascompared to a normal control.
 29. The oligonucleotide of claim 17,wherein the oligonucleotide comprises the sequences set forth as SEQ IDNOS: 15 to
 94. 30. A composition comprising one or more oligonucleotidesspecific for one or more Sirtuin (SIRT) polynucleotides, saidpolynucleotides comprising antisense sequences, complementary sequences,alleles, homologs, isoforms, variants, derivatives, mutants, fragments,or combinations thereof.
 31. The composition of claim 30, wherein theoligonucleotides have at least about 40% sequence identity as comparedto any one of the nucleotide sequences set forth as SEQ ID NOS: 15 to94.
 32. The composition of claim 30, wherein the oligonucleotidescomprise nucleotide sequences set forth as SEQ NOS: 15 to
 94. 33. Thecomposition of claim 32, wherein the oligonucleotides set forth as SEQID NOS: 15 to 94 comprise one or more modifications or substitutions.34. The composition of claim 33, wherein the one or more modificationsare selected from: phosphorothioate, methylphosphonate, peptide nucleicacid, locked nucleic acid (LNA) molecules, and combinations thereof. 35.A method of preventing or treating a disease associated with at leastone Sirtuin (SIRT) polynucleotide and/or at least one encoded productthereof, comprising: administering to a patient a therapeuticallyeffective dose of at least one antisense oligonucleotide that binds to anatural antisense sequence of said at least one Sirtuin (SIRT)polynucleotide and modulates expression of said at least one Sirtuin(SIRT) polynucleotide; thereby preventing or treating the diseaseassociated with the at least one Sirtuin (SIRT) polynucleotide and/or atleast one encoded product thereof.
 36. The method of claim 35, wherein adisease associated with the at least one Sirtuin (SIR) polynucleotide isselected from cancer (e.g., breast cancer, colorectal cancer, CCL, CML,prostate cancer), a neurodegenerative disease or disorder (e.g.,Alzheimer's Disease (AD), Huntington's disease, Parkinson's disease,Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis, and disorderscaused by polyglutamine agwegation); skeletal muscle disease (e.g.,Duchene muscular dystrophy, skeletal muscle atrophy, Becker's dystrophy,or myotonic dystrophy); a metabolic disease or disorder (e.g., insulinresistance, diabetes, type 2 diabetes, obesity, impaired glucosetolerance, metabolic syndrome, adult-onset diabetes, diabeticnephropathy, hyperglycemia, diabetic nephropathy, Hypercholesterolemia,dyslipidemia hyperlipidemia and an age-related metabolic disease etc.),a disease or disorder associated with impaired regulation of insulinlevel, neuropathy (e.g., sensory neuropathy, autonomic neuropathy, motorneuropathy, retinopathy), a disease or disorder associated with aketogenic condition, a disease or disorder associated with impairedenergy homeostasis, a disease or disorder associated with impairedAcetyl-CoA synthetase 2 activity, a disease or disorder associated withmetabolic homeostasis, a lipid metabolism disease or disorder, a diseaseor disorder associated with impaired thermogenesis, a disease ordisorder associated with mitochondrial dysfunction, neuropathy (e.g.,sensory neuropathy, autonomic neuropathy, motor neuropathy,retinopathy), a liver disease (e.g., due to alcohol abuse or hepatitis,fatty liver disease etc.); age-related macular degeneration, bonedisease (e.g., osteoporosis), a blood disease (e.g., a leukemia); liverdisease (e.g., due to alcohol abuse or hepatitis); obesity; boneresorption, age-related macular degeneration, AIDS related dementia,ALS, Bell's Palsy, atherosclerosis, a cardiac disease (e.g., cardiacdysrhymias, chronic congestive heart failure, ischemic stroke, coronaryartery disease and cardiomyopathy), chronically degenerative disease(e.g., cardiac muscle disease), chronic renal failure, type 2 diabetes,ulceration, cataract, presbiopia, glomerulonephritis, Guillan-Barresyndrome, hemorrhagic stroke, rheumatoid arthritis, inflammatory boweldisease, SLE, Crohn's disease, osteoarthritis, osteoporosis, ChronicObstructive Pulmonary Disease (COPD), pneumonia, skin aging, urinaryincontinence, a disease or disorder associated with mitochondrialdysfunction (e.g., mitochondrial myopathy, encephalopathy, Leber'sdisease, Leigh encephalopathia, Pearson's disease, lactic acidosis,‘mitochondrial encephalopathy; lactic acidosis and stroke like symptoms’(MELAS) etc.) and a disease or disorder associated with neuronal celldeath, degenerative syndrome, aging, a disease or disorder associatedwith telomere dysfunction, a disease or disorder associated withimpaired chromatin regulation, a disease or disorder associated withpremature cellular senescence, a disease or disorder associated withimpared SIRT6 mediated DNA repair and a condition characterized byunwanted cell loss.
 37. A method of preventing or treating a skincondition associated with at least one Sirtuin (SIRT) polynucleotideand/or at least one encoded product thereof, comprising: administeringto a patient having a skin condition or at risk of developing a skincondition a therapeutically effective dose of at least one antisenseoligonucleotide that binds to a natural antisense sequence of said atleast one Sirtuin (SIRT) polynucleotide and modulates expression of saidat least one Sirtuin (SIRT) polynucleotide; thereby preventing ortreating the disease skin condition associated with the at least oneSirtuin (SIRT) polynucleotide and/or at least one encoded productthereof.
 38. The method of claim 38, wherein the skin condition iscaused by caused by inflammation, light damage or aging.
 39. The methodof claim 39, wherein the skin condition is the development of wrinkles,contact dermatitis, ample dermatitis, actinic keratosis, keratinizationdisorders, an epidermolysis bullosa disease, exfoliative dermatitis,seborrheic dermatitis, an erythema, discoid lupus erythematosus,dermatomyositis, skin cancer, or an effect of natural aging.